Real-time crispr endonuclease activity assay

Abstract

The invention comprises a rapid in vitro method of assessing activity of a CRISPR Cas9 endonuclease, and a nucleic acid substrate comprising a FRET pair for use in the method. Compositions, diagnostic methods, and kits are also disclosed.

Description

Attorney Docket Number: CBI060.30PATENTREAL-TIME CRISPR ENDONUCLEASE ACTIVITY ASSAYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application serial no. 63 / 687,615 filed on 27 August 2024, and in incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The invention related to the field of nucleic acid-modifying enzymes and more specifically, to the field of developing and testing active endonucleases.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] None.SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on 17 July 2025, is named CBI060 30 SL.xml and is 35,573 bytes in size.BACKGROUND OF THE INVENTION

[0005] CRISPR endonucleases and especially Cas9 have found many uses in life science and clinical applications. Isolating and working with Cas9 requires rapid and convenient methods of assessing endonuclease activity. The most widely used Cas9 assays are cumbersome and low throughput. There are no conventional means to rapidly measure differences in activity between different sources, variants, or lots of Cas9. Furthermore, the commonly used methods are endpoint and not real-time. For example, a typical fluorescence-based assay for measuring Cas9 activity (Seamon et al., (2018) Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity, Anal. Chem. 90(11):6913) cannot be monitored in real time because Cas9 is very slow to dissociate from its cleavage products under the reaction conditions. A denaturing reagent must be added in order to generate a fluorescent signal adding to the complexity of the assay while limiting its throughput and kinetic resolution.Attorney Docket Number: CBI060.30PATENT

[0006] There is an unmet need for a rapid CRISPR Cas9 endonuclease assay that would work in real time. Such an assay would aid the efforts to improve enzyme purification and optimizing CRISPR Cas9-based assays and gene editing protocols.

[0007] SUMMARY OF THE INVENITON

[0008] In one embodiment, the invention is a nucleic acid substrate for detecting activity of a CRISPR Cas9 endonuclease, the substrate comprising: a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer and a protospacer adjacent motif (PAM) for a Cas9; a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9. In some embodiments, the acceptor fluorophore is a quencher fluorophore. In some embodiments, the donor fluorophore and the acceptor fluorophore are on the non-target strand. In some embodiments, one of the donor fluorophore and the acceptor fluorophore is on the non-target strand. In some embodiments, the donor fluorophore and the acceptor fluorophore are situated within the post-cleavage trimming (PCT) zone of the Cas9.

[0009] In some embodiments, the donor fluorophore is selected from a group consisting of 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2', 4', 1,4, -tetrachlorofluorescein (TET), 2', 4', 5', 7', 1 ,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6- carboxyfluorescein (JOE), coumarin dyes, Alexa Fluor dyes, IRDye 800CW, Cascade Blue, Pacific Blue, Pacific Orange, Texas Red, and BODIPY® dyes. In some embodiments, the acceptor fluorophore is selected from a group consisting of tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), DABSYL, DABCYL (4-[[4-(dimethylamino)-phenyl]- azo]-benzoic acid), Cy5 and Cy5.5, anthraquinone dyes, nitrothiazole dyes, nitroimidazole dyes, LC-Red 610, LC-Red 640, LC-Red 705, JA286, DDQ-I, DDQ-II, QSY-7, QSY-21, IRDye QC1, Iowa Black FQ, Iowa Black RQ, HEX (hexachloro-fluorescein), TET (tetrachloro-fluorescein), JOE (5'-Dichloro-dimethoxy-fluorescein), BODIPY® dyes, Eclipse 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-(l-nitro-phenyl)-azo)-yl-((2,5-dimethoxy-phenyl)-azo)]- aniline), and pyridinyl-isoquinoline-dione dyes.Attorney Docket Number: CBI060.30PATENT

[0010] In some embodiments, the protospacer adjacent motif (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'.

[0011] In some embodiments, the substrate further comprises a structure inhibiting cleavage of the substrate by an exonuclease is selected from a hairpin, a strand overhang, and a nucleic acid modification, e.g., one or more phosphorothioate linkages.

[0012] In some embodiments, the non-target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37. In some embodiments, the target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.

[0013] In one embodiment, the invention is a composition for detecting activity of a CRISPR Cas9 endonuclease comprising the nucleic acid substrate described above. In some embodiments, the composition further comprises a Cas9. In some embodiments, composition further comprises a nucleic acid targeting nucleic acid (NATNA). In some embodiments, the NATNA is selected from a single guide and a dual guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a protospacer capable of hybridizing to the spacer in the substrate. In some embodiments, the NATNA comprises DNA and RNA nucleotides.

[0014] In one embodiment, the invention is a method for detecting activity of a CRISPR Cas9 endonuclease comprising: contacting a Cas9 with a reaction mixture comprising a nucleic acid substrate comprising: a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence for the Cas9; a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9; and measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates activity of the Cas9. In some embodiments, the protospacer adjacent motif (PAM) consists 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 reaction mixture further comprises an endonuclease and a nucleic acidAttorney Docket Number: CBI060.30PATENT targeting nucleic acid (NATNA). In some embodiments, the NATNA is selected from a single guide and a dual guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a protospacer capable of hybridizing to the spacer in the substrate. In some embodiments, the NATNA comprises DNA and RNA nucleotides. In some embodiments, the contacting comprises contacting a series of reaction mixtures with the same endonuclease but with one of a series of different NATNA. In some embodiments, the contacting comprises contacting a series of reaction mixtures comprising with the same endonuclease under different reaction conditions. In some embodiments, the contacting comprises contacting a series of reaction mixtures with one of a series of different isolates of the Cas9. In some embodiments, the contacting in step (a) comprises contacting a series of reaction mixtures comprising one of a series of different nucleic acid substrates comprising different sequences with the Cas9. In some embodiments, the substrate comprises a structure inhibiting cleavage of the substrate by an exonuclease is selected from a hairpin, a strand overhang, and a nucleic acid modification, e.g., one or more phosphorothioate linkages. In some embodiments, the nucleic acid substrate or the Cas9 are in an unpurified form. In some embodiments, the non-target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37. In some embodiments, the target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38. In some embodiments, the fluorescence is measured continuously thereby detecting the activity of the CRISPR Cas9 endonuclease in real time.

[0015] In one embodiment, the invention is a kit for detecting activity of a CRISPR Cas9 endonuclease comprising a nucleic acid substrate comprising: a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence for a Cas9; a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9. In some embodiments, kit further comprises a nucleic acid targeting nucleic acid (NATNA) capable of forming a complex with the Cas9. In some embodiments, the NATNA is a CRISPR guide RNA selected from a single guide and a dual guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments,Attorney Docket Number: CBI060.30PATENT the NATNA comprises a protospacer capable of hybridizing to the spacer in the substrate. In some embodiments, the substrate comprises a structure inhibiting cleavage of the substrate by an exonuclease is selected from a hairpin, a strand overhang, and a nucleic acid modification, e.g., one or more phosphorothioate linkages. In some embodiments, the non-target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37. In some embodiments, the target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.

[0016] In one embodiment, the invention is an apparatus for detecting activity of a Cas9 with the substrate of claim 1 comprising: a reaction chamber for performing enzymatic reactions and a fluorescence detector. In some embodiments, the detector is capable of continuously measuring fluorescence thereby detecting the activity of the Cas9 in real time.

[0017] In one embodiment, the inventing is a method for detecting the presence of a target nucleic acid in a sample, the method comprising: contacting a sample with a reaction mixture comprising a Cas9 and a nucleic acid probe capable of hybridizing to a target nucleic acid, the probe comprising: i) a spacer sequence and a protospacer adjacent motif (PAM) sequence for a Cas9; ii) a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9; measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates the presence of the target nucleic acid in the sample. In some embodiments, the 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’s sequence characteristic of a patient’s disease or condition. In some embodiments, the protospacer adjacent motif (PAM) consists 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 reaction mixture further comprises a NATNA selected from a single guide and a dual guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a protospacer region capable of hybridizing to the spacer sequence in the probe. In some embodiments, the NATNA comprises DNA and RNA nucleotides. In some embodiments, the probe comprises a structure inhibiting cleavage of theAttorney Docket Number: CBI060.30PATENT probe by an exonuclease, wherein the structure is selected from a hairpin, a strand overhang, and a nucleic acid modification. In some embodiments, the nucleic acid modification comprises one or more phosphorothioate linkages. In some embodiments, the non-target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37. In some embodiments, the target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.

[0018] In one embodiment, the invention is a kit for performing a diagnostic procedure according to the method of claim 46 comprising a probe comprising: i) a spacer sequence and a protospacer adjacent motif (PAM) sequence for a Cas9; ii) a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9. In some embodiments, the kit further comprises the Cas9. In some embodiments, the kit further comprises a nucleic acid targeting nucleic acid (NATNA) capable of forming a complex with the Cas9. In some embodiments, the NATNA is selected from a single guide and a dual guide. In some embodiments, the NATNA comprises a crRNA and a tracrRNA. In some embodiments, the NATNA comprises a protospacer region capable of hybridizing to the spacer in the probe. In some embodiments, the NATNA comprises DNA and RNA nucleotides— In some embodiments, the probe comprises a structure inhibiting cleavage by an exonuclease is selected from a hairpin, a strand overhang, and a nucleic acid modification, e.g., or more phosphorothioate linkages. In some embodiments, the non-target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37. In some embodiments, the target strand of the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGURE 1 is a diagram of the post-cleavage trimming (PCT) property of Cas9 described in Stephenson et al. (2018).

[0020] FIGURE 2 is a diagram of one example of the Cas9 substrate according to the invention.Attorney Docket Number: CBI060.30PATENT

[0021] FIGURE 3 shows fluorescence measurement according to the method of the invention indicating Cas9 activity (substrate: TRAC gene sequence).

[0022] FIGURE 4 illustrates assay linearity with respect to guide RNA (chRDNA) concentration.

[0023] FIGURE 5 illustrates assay linearity with respect to DNA substrate concentration.

[0024] FIGURE 6 illustrates assay linearity with respect to nucleoprotein complex concentration (Cas9 complexed with chRDNA and ch-acr).

[0025] FIGURE 7 illustrates assay linearity with respect to guide RNA (chRDNA) concentration.

[0026] FIGURE 8 illustrates assay linearity with respect to DNA-containing tracrRNA (ch-acr) concentration.

[0027] FIGURE 9 illustrates a comparison of two substrates designed according to the method of the invention.

[0028] FIGURE 10 shows fluorescence measurement according to the method of the invention indicating Cas9 activity (substrate: PDCD1 gene sequence).DETAILED DESCRIPTION OF THE INVENTION

[0029] The following definitions are provided to aid in understanding of the disclosure.

[0030] The term “endonuclease” refers to an enzyme catalyzing the hydrolysis of a phosphodiester bond between two nucleoside residues within a polynucleotide (DNA or RNA) wherein neither nucleoside residue is a terminal one.

[0031] The term “CRISPR repeat” or “CRISPR repeat sequence” refers to a minimum CRISPR repeat sequence.

[0032] The term “inhibiting” refers to the ability of a chemical structure to partially or completely inhibit a chemical reaction. A skilled artisan would understand that whether the inhibition is partial or complete depends on the sensitivity of detection methods. The term “inhibiting cleavage” with respect to a nuclease refers to the ability to detectably diminish the amount of cleavage product. The term “preventing cleavage” with respect to a nuclease refers to the ability to diminish the amount of cleavage product below the level of detection.

[0033] The term “NATNA” refers to a nucleic acid targeting nucleic acid. NATNA may be a part of the programmable endonuclease system, such as a CRISPR system. NATNA may beAttorney Docket Number: CBI060.30PATENT comprised of two nucleic acid targeting polynucleotides (“dual guide”) including a CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). NATNA may be comprised of an engineered single nucleic acid targeting polynucleotide (“single guide”) comprising crRNA and tracrRNA connected by a fusion region (linker). The crRNA may comprise a targeting region and an activating region. The tracrRNA may comprise a region capable of hybridizing to the activating region of the crRNA. The term “targeting region” refers to a region that is capable of hybridizing to a sequence in a target nucleic acid. The term “activating region” refers to a region that interacts with a polypeptide, e.g., a CRISPR nuclease.

[0034] The terms “recognition sequence” and “recognition site” refer to a sequence of nucleotides in a nucleic acid preferentially or exclusively recognized by an endonuclease resulting in binding of the endonuclease to the nucleic acid. In some but not all cases, a recognition sequence or recognition site also include a cleavage sequence or cleavage site.

[0035] The terms “cleavage sequence” and “cleavage site” refer to a sequence of nucleotides in a nucleic acid preferentially or exclusively cleaved by an endonuclease. In some but not all cases, a cleavage site is the same as a recognition site. In some but not all cases, a cleavage site is different from a recognition site. In such cases, the recognition site must also be present in the nucleic acid for cleavage of the cleavage site to occur.

[0036] The term “post-cleavage trimming” refers to bi-directional degradation of cleavage products catalyzed by CRISPR / Cas9 nuclease and comprising removal of up to 10 nucleotides from the 3’-end of the PAM-distal fragment of the non-target strand and removal of a single nucleotide from the 5 ’-end of the PAM-proximal fragment of the non-target strand. See Stephenson et al., (2018) Bi-directional degradation of cleavage products catalyzed by CR1SPR / Cas9, J. Am. Chem. Soc. 140:3743.

[0037] Cas9 is the first to be discovered CRISPR endonuclease that has been used for gene editing in agriculture, life sciences and medical applications including diagnostic and therapeutic applications. A rapid and convenient method of assessing Cas9 activity can be used to validate the assay, the quality of each of the reagents and the final products of the reaction. The most widely used Cas9 assays are cumbersome, low-throughput and end-point (not real-time). For example, a fluorescence-based assay for measuring Cas9 activity (Seamon et al., (2018) Versatile High- Throughput Fluorescence Assay for Monitoring Cas9 Activity, Anal. Chem. 90(11):6913) cannot be read in real time because Cas9 is very slow to dissociate from its cleavage products under theAttorney Docket Number: CBI060.30PATENT reaction conditions. Disclosed herein are compositions and methods for a rapid fluorescence-based assay for Cas9 activity that can be read in real time.

[0038] 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. Especially popular are probes and substrates labeled with two fluorophores forming a FRET pair. Popular types of dual-labeled probes include Taqman and Molecular Beacon probes.

[0039] In the Taqman assay (U.S. Patent No. 5,210,015), a dual-labeled oligonucleotide probe hybridizes to the nascent amplification product during PCR. Fluorescence is detected when the 5 ’-3’ exonuclease activity of the DNA polymerase hydrolyzes the probe between the two fluorophores.

[0040] Molecular Beacons are hairpin-shaped dual-labeled probes where binding of a probe to its target causes unraveling of the hairpin. The unraveling separates the FRET pair allowing fluorescence of the donor fluorophore to be detected. Tyagi S, et al., (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 14(3):303.

[0041] Nucleic acid substrates labeled with two fluorophores forming a FRET pair and capable of conditional fluorescence can be used to detect in vitro enzymatic activity. Enzymatic activity may be indicative for example, of the presence of an infectious agent in the sample. The use of probes labeled with a fluorophore and a quencher fluorophore to detect microbial contamination as described in U.S. Patent No. 10,663,459. According to this method, the presence of a microorganism in the sample results in cleavage of the probe with one or more nucleases (e.g., endonucleases or exonucleases). All possible cleavages result in physical separation of the fluorophore and the quencher, and emission of detectable fluorescence which is indicative of the presence of a microorganism in the sample.

[0042] A similar principle is employed in the U.S. Patent No. 10,653,800 where an RNA substrate incorporates 2’-O-methyl-modified pyrimidines and is uniquely sensitive to mycoplasmal RNase. The substrate is labeled with a fluorophore and a quencher. The presence of mycoplasma in the sample results in digestion of the probe with the mycoplasmal RNase and emission of detectable fluorescence which is indicative of the presence of mycoplasma in the sample.

[0043] Double-stranded nucleic acid substrates labeled with a fluorophore and a quencher have also been used to detect specific editing activity of CRISPR endonucleases. For example,Attorney Docket Number: CBI060.30PATENTSmith et al., (2020) Probing CRISPR-Casl2a Nuclease Activity Using Double-Stranded DNA- Templated Fluorescent Substrates, Biochemistry 59:1474, describe cleavage of such a substrate with the trans-cleavage (“trans-shredding”) activity of the CRISPR Cast 2a nuclease. The transshredding activity is triggered by binding of the Casl2a-crRNA complex to a double- stranded DNA target. The trans-shredding nuclease activity is directed to any double-stranded DNA in the vicinity of the Casl2a-crRNA-target complex. The shredding of the substrate labeled with a fluorophore and a quencher result in emission of detectable fluorescence which is indicative of the formation of the Casl2a-crRNA-target complex in the sample.

[0044] Activity of CRISPR-Cas nucleases such as Cas9 is typically measured by incubation of the ribonucleoprotein complex (RNP) with a model substrate followed by agarose gel electrophoresis to resolve the cleaved fragments from the intact substrate. This approach is low-throughput and is generally limited to end-point analysis, and thus provides little or no information about the kinetics of the reactions.

[0045] A fluorescence-based assay has also been described for Casl2a, however it measures only the non-specific trans activity of the Casl2a enzyme, (see Smith et al., Biochemistry. 2020 supra). When the Casl2a RNP engages and cleaves the target sequence (cis activity) the Casl2a is activated to non-specifically degrade short fragments of DNA (trans activity or “trans shredding” activity). There are several limitations to measuring trans activity as an indicator of cis activity. First, the exact correlation between the cis and the trans activity is not known. Second, the surrogate assay provides limited kinetic information about the cis activity because the two reactions are performed by the same enzyme and cannot be decoupled. Lastly, the assay lacks general applicability as many endonucleases including some CRISPR endonucleases such as Cas9 to not exhibit trans activity.

[0046] Various amplification-based CRISPR Cas9 nuclease activity assays have been devised. These assays are still unable to monitor the reaction in real time. Zhang et al., (2016) Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification, Chem. Sci. 7:4951) discloses NTEXPAR, a fluorescence-based isothermal amplification method that detects double-stranded DNA cleavage by Cas9. The products of amplification are detected by gel electrophoresis after completion of the amplification reaction. The assay detects Cas9 with high sensitivity but not in real time. Raper et al. disclose an end-pointAttorney Docket Number: CBI060.30PATENT assay utilizing radiolabeled DNA substrates. Raper et al., (2018) Functional insights revealed by the kinetic mechanism of CRISPR / Cas9, J. Am. Chem. Soc. 140:2971.

[0047] A real-time endonuclease cleavage assay has been disclosed in the International Patent Application Pub. No. WO2023076857 Exonuclease-coupled real-time endonuclease activity assay. That assay is generally applicable to site-specific endonucleases and relies on endonuclease cleavage and subsequent exonuclease cleavage to separate a quencher fluorophore from a reporter fluorophore thereby releasing fluorescence. The endonuclease assay described herein is an improved and simplified real-time endonuclease activity assay relying on a unique property of the CRISPR Cas9 endonuclease.

[0048] The invention further comprises a diagnostic assay relying on the sequence-specific activity of the Cas9 endonuclease to indicate the presence of a diagnostic target in a sample.

[0049] As stated above, one existing fluorescence assay for measuring Cas9 activity (Seamon et al., (2018)) requires denaturing the DNA substrate after endonuclease cleavage because of the inconvenient property of Cas9 to remain bound to the substrate after cleavage. It was also discovered that Cas9 possesses a bidirectional trimming activity termed “post-cleavage trimming” (PCT), see Stephenson et al., (2018) Bidirectional degradation of DNA cleavage products catalyzed by CRISPRCas9 J. Am. Chem. Soc. 140(10):3743. The activity is illustrated in FIGURE 1 and includes 3’-5’ degradation of the non-target strand up to the (-10) position on the P AM-distal nucleic acid cleavage product, and a single base removal from the 5 ’-end of the same strand on the P AM-proximal nucleic acid cleavage product. Notably, this enzymatic activity is kinetically significant when compared to extremely slow cleavage product release by Cas9.

[0050] In some embodiments, the invention is a substrate molecule for detecting and assessing the activity of a CRISPR Cas9 endonuclease. In some embodiments, the substrate is a nucleic acid having at least one double stranded region. In some embodiments, the substrate is single stranded but becomes suitable for cleavage by a Cas9 endonuclease when combined with a complementary strand present in the reaction mixture. In some embodiments, the substate is a double-stranded nucleic acid comprising two nucleic acid strands forming a duplex via hybridization. In other embodiments, the substrate is a nucleic acid comprising at least one doublestranded region formed by a single nucleic acid strand forming a secondary structure e.g., a hairpin or a stem-loop structure. One of skill in the art would recognize that hybridization and formation of a double-stranded region do not require 100% complementarity throughout the length of theAttorney Docket Number: CBI060.30PATENT nucleic acid strands forming the double stranded region. Under suitable conditions defined by ionic power of the buffer and temperature, a stable hybrid (a double stranded region) can form between nucleic acid strands with less than 100%, complementarity e.g., 90%, 80%, 75% or less complementarity.

[0051] As illustrated in FIGURE 2, the nucleic acid substrate comprises a target strand comprising a target sequence capable of hybridizing with the protospacer present in the guide nucleic acid (NATNA) guiding the Cas9 endonuclease to the cleavage site. The substrate further comprises a non-target strand comprising a spacer sequence matching the protospacer in the NATNA). It is understood that perfect complementarity between the protospacer and the spacer is not required for nucleic acid hybridization and subsequence cleavage by Cas9. A certain number of mismatches between the spacer can be tolerated, and a maximum number of mismatches can experimentally determined. The non-target strand further comprises a protospacer adjacent motif (PAM) recognized by Cas9. 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’, 5’-TTN-3’, 5’-TTTN-3’ and 5’-TTTV-3’.

[0052] The substrate further comprises a donor fluorophore and an acceptor fluorophore said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair. In some embodiments, the donor is a reporter fluorophore, and the acceptor is a quencher fluorophore such that no fluorescence occurs as long as the reporter and the quencher remain in close proximity. According to the method disclosed herein, the reporter and the quencher become separated via the post-cleavage trimming (PCT) activity of Cas9. Because the PCT zone of Cas9 comprises only the non-target strand, at least one of the reporter and the quencher must be located on the non-target strand. In some embodiments, both the reporter and the quencher are located on the non-target strand. At least one of the reporter and the quencher must be located in the PCT zone of the non- target strand. In some embodiments, both the reporter and the quencher are located in the PCT zone of the non-target strand. One of the advantages of the placing both the reporter and the quencher within the PCT zone is that it minimizes background signal because the fluorophore and quencher are in very close proximity within the short PCT zone. It also avoids any steric effects that would likely occur if the fluorophore quencher pair were incorporated near the cut site, for instance with the fluorophore on one side of the cut-site and the quencher on the other.Attorney Docket Number: CBI060.30PATENT

[0053] In some embodiments, at least one of the reporter and the quencher is conjugated to a nucleotide selected from nucleotides (-1) through (-10) on the PAM-distal fragment of the non-target strand. In some embodiments, at least one of the reporter and the quencher is conjugated to a nucleotide selected from nucleotides (-1) through (-5) on the PAM-distal fragment of the nontarget strand. In some embodiments, the reporter or the quencher is conjugated to the 5 ’-terminal nucleotide of the PAM-proximal fragment of the non-target strand. In some embodiments, one of the reporter and the quencher fluorophores is located on the target strand. In such embodiments, the fluorophore on the target strand is not limited to a specific nucleotide but is placed close enough to the other fluorophore located in the PCT zone to ensure a FRET process between the fluorophores.

[0054] FIGURE 2 illustrates an example where both the reporter and the quencher fluorophore are located in the PCT zone of the PAM-distal portion of the non-target strand. According to the method of the invention, when the substrate is contacted with Cas9, the HNH and the RuvC domains of Cas9 cleave the target strand and the non-target strand respectively three nucleotides away from the PAM. Following the initial cleavage, the RuvC domain of Cas9 effects its PCT activity and removes up to 10 nucleotides from the 3’-end of the PAM-distal fragment of the non-target strand and a single nucleotide from the 5 ’-end of the PAM-proximal fragment of the non-target strand. In the exemplary substrate design shown in FIGURE 2, both fluorophores are located in the PAM-distal portion of the non-target strand. The reporter and the quencher become separated via the PCT activity and detectable fluorescence is emitted.

[0055] Fluorescence resonance energy transfer (FRET), also known as Foerster (or Forster) resonance energy transfer is transfer of excitation energy from one molecule to another without fluorescence and re-absorption. A donor chromophore enters an electronically excited state after having absorbed light of a certain wavelength. The donor transfers energy to an acceptor and the acceptor is promoted to its electronically excited state. Subsequently, the electronically excited state of the acceptor decays so that in turn detectable light is emitted. Because the acceptor diminishes or quenches fluorescence of the donor, the acceptor is sometimes referred to as a quencher. The donor is sometimes referred to as a reporter. In conventional FRET technology donor and acceptor are both fluorophores. The donor fluorophore absorbs the light of a certain absorption wavelength and the acceptor emitted light of a particular emission wavelength which is longer than the absorption wavelength. FRET occurs when donor and acceptor are in closeAttorney Docket Number: CBI060.30PATENT proximity (e.g., 1-10 nm). In some embodiments, the donor fluorophore and the acceptor fluorophore are placed between 0 and 12 nucleotides apart. The donor fluorophore and the acceptor fluorophore are placed on the same strand of the substrate or on different (opposite) strands of the substrate. Either the donor, the acceptor or both the donor and the acceptor can be placed near a terminus of a nucleic acid strand, e.g., a 5 ’-terminus or a 3 ’-terminus. The donor and the acceptor fluorophores may be on the same strand or on opposite strands. A skilled practitioner would recognize various options for placing the donor fluorophore and the acceptor fluorophore (or the reporter fluorophore and the quencher) within the double- stranded substrate so that the desired proximity of the fluorophores (e.g., 1-10 nm) is achieved.

[0056] Existing literature provides ample guidance for selecting appropriate reporterquencher pairs capable of FRET, see e.g., U.S. Patent Nos. 5,538,848; 8,350,038; and 8,137,616. Generally, as recommended in U.S. Patent Application Publication US20060088855, the donor fluorophores absorb in the range of 350-800 nm, preferably 350-600 nm or 500-750 nm and the distance between donor and acceptor be 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); and the like.

[0057] Many donors, acceptors and donor / acceptor pairs that exhibit FRET phenomenon are commercially available. Popular donors include fluorescein dyes such as 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2', 4', 1,4, -tetrachlorofluorescein (TET), 2',4',5',7', 1,4- hexachlorofluorescein (HEX), and 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE). Other donors include coumarin dyes, Alexa Fluor family of dyes, IRDye 800CW, Cascade Blue, Pacific Blue, Pacific Orange, and Texas Red. Popular acceptors include rhodamine dyes such as tetramethyl-6-carboxyrhodamine (TAMRA), and tetrapropano-6-carboxyrhodamine (ROX), DABSYL, DABCYL, cyanine dyes including Cy5 and Cy5.5, anthraquinone, nitrothiazole, and nitroimidazole compounds. Additional acceptors are LC-Red 610, LC-Red 640, LC-Red 705, JA286, DDQ-I, DDQ-II, QSY-7, QSY-21, IRDye QC1, Iowa Black FQ and Iowa Black RQ. andAttorney Docket Number: CBI060.30PATENT sulfonated cyanine dyes disclosed in U.S. Patent No. 6,027,709. Popular donor-acceptor combinations include fluorescein / rhodamine, especially carboxyfluorescein / tetramethyl- rhodamine (FAM / TAMRA). TAMRA as quencher can also be paired with such donors as HEX (hexachloro-fluorescein), TET (tetrachloro-fluorescein), JOE (5'-Dichloro-dimethoxy- fluorescein) and cyanine dyes. Another donor / acceptor pair is disclosed in the U.S. Patent No. 9,796,746 and is composed of an oxidized form of a carbaNADH-based first fluorophore and a second fluorophore that is excitable with light having a wavelength of 445-540 nm and an emission maximum greater than 560 nm.

[0058] Another group of fluorescent compounds are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are 1- dimethyl-aminonaphthyl-5-sulfonate, l-anilino-8-naphthalene sulfonate and 2-p-touidinyl-6- naphthalene sulfonate. 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.

[0059] Another category of fluorophores are the BODIPY® dyes described in U.S. Patent No. 5,994,063. "BODIPY®" refers to a class of modified, spectrally-discriminating fluorophores wherein the parent heterocyclic molecule is a dipyrrometheneboron difluoride compound. Most BODIPY® fluorophores have adsorption maxima of about 450 to 700, and emission maxima of about 450 to 700. Examples include BODIPY® 503 / 512-SE (4,4-difluoro-5,7-dimethyl-4-bora- 3a,4a-diaza-s-indacene-3-propionicacid), BODIPY®523 / 547 (4,4-difluoro-5-phenyl-4-bora- 3a,4a-diaza-s-indacene-3-propionic acid), BODIPY® 530 / 550 (4,4-difluoro-5,7-diphenyl-4-bora- 3 a, 4a-diaza-s-indacene-3 -propionic acid), BODIPY® 558 / 568 (4,4-difluoro-5-(2-thienyl)-4bora- 3a,4a-diaza-s-indacene-3-propionic acid), BODIPY® 564 / 570 (4,4-difluoro-5-styryl-4-bora- 3 a, 4a-diaza-s-indacene-3 -propionicacid), BODIPY® 576 / 589 (4,4-difluoro-5-(2-pyrrolyl)-4- bora-3 a, 4a-diaza-s-indacene-3 -propionic acid), and BODIPY® 581 / 591 (4,4-difluoro-5-(4- phenyl-l,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

[0060] One type of quenchers are “dark quenchers.” These non-fluorescent acceptors enable low background fluorescence and thus improve assay sensitivity. When a dark quencher is used, the donor fluorophore does not emit light until the quencher is removed from the proximity of the donor. For example, if donor and quencher are conjugated to an oligonucleotide, fluorescence of the donor may occur only when the quencher is removed though hydrolysis of theAttorney Docket Number: CBI060.30PATENT oligonucleotide by a nuclease. One example of a dark quencher is DABCYL (4-[[4- (dimethylamino)-phenyl]-azo]-benzoic acid) which quenches donor dyes in a range of from 380 to 530 nm. Another dark quencher is Eclipse Quencher (4-[[2-chloro-4-nitro-phenyl]-azo]-aniline (available from Epoch Biosciences, Inc.) which has an absorption maximum at 530 nm and efficiently quenches over a spectrum from 520 to 670 nm. Yet another category of dark quenchers is the 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-(l-nitro-phenyl)-azo)-yl-((2,5-dimethoxy- phenyl)-azo)] -aniline) (all available from Biosearch Technologies, Inc.).

[0061] Another type of quencher includes the pyridinyl-isoquinoline-dione derivatives disclosed in the U.S. Patent No. 8,350,038. These compounds feature a low background signal and high quenching efficiency. Yet another category of quenchers is the non-fluorescent cyanine quencher compounds attached to base of a nucleotide via a linker compound disclosed in the U.S. Patent No. 6,348,596. Yet another category of quenchers is the weakly luminescent cyanines that are substituted by one or more heteroaromatic quenching moi eties disclosed in the U.S. Patent No. 8,093,411. These quenchers exhibit little or no observable luminescence and efficiently quench a broad spectrum of luminescent compounds.

[0062] Methods of synthesizing oligonucleotides and of covalently attaching fluorophores to nucleic acids are known in the art. See e.g., U.S. Patent Nos. 3,996,345; 4,351,760; 4,757,141; 4,739,044; 4,997,928; 5,538,848; 5,188,934; 5,231,191; and 7,759,469; 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 etal., Nucleic Acids Research, 15:4837 (1987) (5' mercapto group); Nelson et al., Nucleic Acids Research, 17:7187-7194 (1989) (3' amino group). For synthesizing labeled nucleic acid probes, functional groups and linking moieties may be used. As commonly used, pre-synthesized fluorophore-labeled nucleotides are incorporated into an oligonucleotide using standard phosphoramidite-based chemistry. By incorporating such nucleotides into a desired position in an oligonucleotide, donor and acceptor fluorophores may be incorporated at any internal or terminal position in the oligonucleotide. In the pre-synthesized fluorophore-labeled nucleotides the label may be bound by a functional group attached forAttorney Docket Number: CBI060.30PATENT example, to an amino group of a nucleotide’s base. In other embodiments, the label is attached to a part of a nucleotide via a linking moiety. For example, in some embodiments, the nucleotide base is modified to allow conjugation to a label. For example, U.S. Patent No. 7,759,469 discloses substituted nitroindole nucleotides that can be conjugated to a fluorophore.

[0063] In some embodiments, the double stranded nucleic acid substrate or the doublestranded portion of the nucleic acid substrate is between about 10 and about 90 base pairs in length, e. ., between 35 and 90 base pairs long and comprises a Cas9 PAM (5 nt), and a spacer (20 nt) (FIGURE 2)

[0064] In some embodiments, the nucleic acid substrate comprises a duplex combining the corresponding “top” and “bottom” strands of SEQ ID NOs: 1-38. For illustration purposes only, the nucleic acid strands are arbitrarily designated “top” and “bottom” to indicate complementary strands forming a duplex. In some embodiments, the nucleic acid substrate comprises the fluorophore-bearing strand selected from SEQ ID NOs: 1-38, to be paired with a target nucleic acid found in the test sample. One of skill in the art would recognize that SEQ ID NOs: 1-38 represent merely examples of fluorophore and quencher placement within the substrate. All other fluorophore and quencher placements that fall under the recommendations described herein are considered equivalents of SEQ ID NOs: 1-38 that perform essentially the same function in essentially the same way.

[0065] In some embodiments, the nucleic acid substrate of the invention includes chemical modifications. In some embodiments, the modification results in increased stability of the nucleic acid duplex. In some embodiments, the modification confers resistance to nuclease digestion or inhibits nucleases present in the reaction mixture. For example, in some embodiments, the modification confers resistance to exonuclease digestion or inhibits exonucleases present in the reaction mixture.

[0066] 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 heteroatom intemucleoside linkages.

[0067] Another type of backbone modification is modification of a sugar moiety. In some embodiments, the modification involves incorporation of a 6-membered morpholino ring in place of a ribose or deoxyribose ring. Another backbone modification involves incorporation of a cyclohexenyl ring in place of a ribose or deoxyribose (ceNA). Yet another backbone modificationAttorney Docket Number: CBI060.30PATENT involves incorporation of Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 4' carbon atom of the ribose thereby forming a bicyclic structure having a 2'-C,4'-C- oxymethylene linkage. LNAs are characterized by duplex stability and resistance to 3 ’-5’ exonuclease digestion.

[0068] In some embodiments, the modification is a nitrogenous base modification. For example, the double-stranded nucleic substrate may incorporate one or more 5 -methylcytosine (5- me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl ( — C=C — CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5 -substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3 -deazaguanine and 3 -deazaadenine. Modified nucleobases can include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin-2(3H)- one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin- 2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (Hpyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one), 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Nucleobases can be useful for increasing the binding affinity of a polynucleotide compound. These can include 5-substituted pyrimidines, 6-azapyrimidines and N- 2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions can increase nucleic acid duplex stability by 0.6- 1.2° C. and can be suitable base substitutions (e g., when combined with 2'-O-methoxyethyl sugar modifications).

[0069] One of skill in the art will appreciate that nucleic acid modifications should not be included in the recognition site or the cleavage site for CRISPR Cas9 or in the PCT zone if the modification is capable of interfering with the endonuclease activity or with the PCT activity of Cas9.Attorney Docket Number: CBI060.30PATENT

[0070] As can be seen in FIGURE 2, the substrate comprises a CRISPR Cas9 recognition sequence (or a recognition site) and CRISPR Cas9 cleavage site.

[0071] The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus is found many prokaryotic genomes and provides resistance to invasion of foreign nucleic acids. Structure, nomenclature, and classification of CRISPR loci are reviewed in Makarova et al., (2011) Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 9(6): 467-477 and Koonin et al., (2023) Discovery of diverse CRISPR-Cas systems and expansion of the genome engineering toolbox, Biochemistry 62:3465-3487.

[0072] Briefly, a typical CRISPR locus includes a number of short repeats regularly interspaced with spacers. The CRISPR locus also includes coding sequences for CRISPR- associated (Cas) genes. A spacer-repeat sequence unit encodes a crisprRNA (crRNA). In vivo, a mature crRNAs is processed from a polycistronic transcript referred to as pre-crRNA or pre- crRNA array. The repeats in the pre-crRNA array are recognized by Cas-encoded proteins that bind to and cleave the repeats liberating mature crRNAs. CRISPR systems perform cleavage of a target nucleic acid wherein Cas proteins and crRNA form a CRISPR ribonucleoproteins (crRNP). The crRNA molecule guides the crRNP to the target nucleic acid (e.g., a foreign nucleic acid invading a bacterial cell) and the Cas nuclease proteins cleave the target nucleic acid.

[0073] Class 2, Type II CRISPR systems to which Cas9 belongs include a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to a crRNA repeat in the pre-crRNA array and recruits endogenous RNaselll to cleave the pre-crRNA array. The tracrRNA / crRNA complex can associate with Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. Hybridization of the crRNA to the target nucleic acid activates the Cas9 nuclease for target nucleic acid cleavage.

[0074] As shown in FIGURE 2, the substrate comprises a recognition sequence (or a recognition site) for Cas9 including a protospacer adjacent motif (PAM). 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', 5’-TTN-3’, 5’-TTTN-3’ and 5’- TTTV-3’. The substrate further comprises a Cas9 cleavage site which is three nucleotides away from the PAM on both the target and the non-target strands.

[0075] The recognition sequence for Cas9 in the substrate further comprises a NATNA binding site (“spacer”). CRISPR nucleases including Cas9 do not cleave a fixed recognitionAttorney Docket Number: CBI060.30PATENT sequence but instead are guided by a nucleic acid guide termed “guide RNA” and termed herein “a nucleic acid targeting nucleic acid (NATNA).” The NATNA comprises a “protospacer” sequence complementary to the “spacer” in the Cas9 recognition site on the substrate (FIGURE 2).

[0076] The reaction mixture that enables cleavage by Cas9 further requires the presence of a nucleic acid targeting nucleic acid (NATNA) comprising tracrRNA and crRNA.

[0077] In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, a NATNA can comprise, in the order of 5' to 3', a spacer extension, a spacer, a minimum CRISPR repeat, a single guide connector, a minimum tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some instances, a nucleic acid-targeting nucleic acid can comprise, a tracrRNA extension, a 3' tracrRNA sequence, a minimum tracrRNA, a single guide connector, a minimum CRISPR repeat, a spacer, and a spacer extension in any order.

[0078] In some embodiments, the guide nucleic acid-targeting nucleic acid can comprise a single guide NATNA. The NATNA comprises a spacer sequence which can be engineered to hybridize to the target nucleic acid sequence. The NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to a tracrRNA sequence. Optionally, NATNA can have a spacer extension and a tracrRNA extension. These elements can include elements that can contribute to stability of NATNA. The CRISPR repeat and the tracrRNA sequence can interact, to form a base-paired, double-stranded structure. The structure can facilitate binding of the endonuclease to the NATNA.

[0079] In some embodiments, the single guide NATNA comprises a spacer sequence located 5' of a first duplex which comprises a region of hybridization between a minimum CRISPR repeat and minimum tracrRNA sequence. The first duplex can be interrupted by a bulge. The bulge facilitates recruitment of the endonuclease to the NATNA. The bulge can be followed by a first stem comprising a linker connecting the minimum CRISPR repeat and the minimum tracrRNA sequence. The last paired nucleotide at the 3' end of the first duplex can be connected to a second linker connecting the first duplex to a mid-tracrRNA. The mid-tracrRNA can comprise one or more additional hairpins.

[0080] In some embodiments, the NATNA can comprise a double guide (or dual guide) nucleic acid structure. The double guide NATNA comprises a spacer extension, a spacer, a minimum CRISPR repeat, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and aAttorney Docket Number: CBI060.30PATENT tracrRNA extension. The double guide NATNA does not include the single guide connector. Instead, the minimum CRISPR repeat sequence comprises a 3' CRISPR repeat sequence and the minimum tracrRNA sequence comprises a 5' tracrRNA sequence and the double guide NATNAs can hybridize via the minimum CRISPR repeat and the minimum tracrRNA sequence.

[0081] In some embodiments, the NATNA is an engineered nucleic acid comprising one or more DNA residues along with the RNA residues. DNA-containing crRNA (CRISPR hybrid R(D)NA or chRDNA) and DNA-containing tracrRNA (chimeric activating CRISPR RNA or ch- acr) and their properties have been described in Donohoue et al., (2021) Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells, Mol. Cell 81 :3637. 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 (Cas9) may be composed of two strands forming a secondary structure that includes an activating region composed of an upper duplex region, a lower duplex region, a bulge, a targeting region, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. Other chRDNA may be a single guide R(D)NA for use with a Type II CRISPR system (Cas9) comprising a targeting region, and an activating region composed of and a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. For example, the targeting region may comprise DNA or a mixture of DNA and RNA, and an activating region may comprise RNA or a mixture of DNA and RNA.

[0082] In some embodiments, the guide RNA includes nucleic acid modifications, e.g., the modifications conferring resistance to ribonucleases. This feature is especially advantageous in crude lysate assays described below.

[0083] In some embodiments, the invention is a method for detecting activity of a CRISPR Cas9 endonuclease using the substrate described herein. To enable activity of Cas9, a NATNA is also utilized in the method. The method comprises contacting a Cas9 to be tested with a reaction mixture including a double-stranded nucleic acid substrate comprising: a spacer for binding of a NATNA, a PAM suitable for Cas9, a donor fluorophore and an acceptor fluorophore (or a reporter fluorophore and a quencher fluorophore), wherein at least one of the donor fluorophore and an acceptor fluorophore located in the post-trimming cleavage (PCT) zone for Cas9, the fluorophoresAttorney Docket Number: CBI060.30PATENT forming a Fluorescence Resonance Energy Transfer (FRET) pair (FIGURE 2). The reaction mixture is incubated under the conditions suitable for Cas9 cleavage of its cleavage site and for the PCT activity of Cas9. In some embodiments, such conditions include {20 mM HEPES:KOH pH 7.5, 100 mM KC1, 5 mM MgC12, 5% glycerol, 100 nM substrate, 150 mM NATNA, 75 nM Cas9, 37°C for approximately 5 to 30 minutes. The method further comprises measuring fluorescence emitted by the reaction mixture. In some embodiments, the fluorescence measurement occurs repeatedly. In some embodiments, the fluorescence measurement occurs continuously. With repeated or continuous measurement of fluorescent activity the method allows real-time assessment of Cas9 activity.

[0084] In some embodiments, the method includes screening, testing, or comparing several preparations of a Cas9 endonuclease. In these embodiments, the method comprises contacting a series of reaction mixtures comprising the same ingredients with a series of different Cas9 endonucleases. In some embodiments, Cas9 is the Streptococcus pyogenes Cas9 (5 / ?yCas9). In some embodiments, Cas9 or a Cas9 homolog is isolated from a bacterial species selected from Staphylococcus aureus, Eubacterium sp. AM18-10LB-B, Neissseria meningitidis, Streptococcus thermophilus, Francisella tularensis (novicida), Actinobacillus succinogenes, Brevibacillus laterosporus, and Campylobacter jejuni. In some embodiments, the Cas9 is a mutant or an engineered variant of the wild-type Cas9 that retains its PCT activity.

[0085] The different preparations may be different isolates of Cas9. In some embodiments, the different preparations are elution aliquots from a chromatography procedure aimed at isolating the Cas9. In some embodiments, invention comprises a method of monitoring elution of Cas9 by performing the endonuclease activity assay described herein on emerging elution fractions from a chromatography procedure and retaining the elution fractions with the highest activity of Cas9. In some embodiments, the different preparations are different “stability samples” of Cas9, i.e., aliquots of Cas9 (or the nucleoprotein complex comprising the Cas9 and a NATNA) stored at different temperature conditions or for different lengths of time e.g., to imitate expected real-life situations encountered by a commercial product. In some embodiments, the invention comprises a method of performing a stability study of a Cas9 (or a Cas9 / NATNA composition) by performing the endonuclease activity assay described herein on a series of Cas9 stability samples to determine a range of acceptable storage conditions and expiration times for Cas9 or a Cas9 / NATNA composition.Attorney Docket Number: CBI060.30PATENT

[0086] In some embodiments, the method includes screening, testing, or comparing several NATNAs capable of binding Cas9 and enabling substrate cleavage and post-trimming activity of Cas9. In these embodiments, the method comprises contacting a series of reaction mixtures comprising the same ingredients, including the same Cas9, with a series of different NATNAs. In some embodiments, the different NATNAs are different “stability samples” i.e., aliquots of NATNA (or the nucleoprotein complex comprising the NATNA and a Cas9) stored at different temperature conditions or for different lengths of time e.g., to imitate expected real-life situations encountered by a commercial product. In some embodiments, the invention comprises a method of performing a stability study of a NATNA (or a NATNA / Cas9 composition) by performing the endonuclease activity assay described herein on a series of NATNA stability samples to determine a range of acceptable storage conditions and expiration times for NATNA or a NATNA / Cas9 composition.

[0087] In some embodiments, the method includes screening, testing, or comparing several nucleic acid sequences in order to identify a preferred or optimal target sequence for Cas9. In these embodiments, the method comprises contacting a series of reaction mixtures comprising the same ingredients including the same Cas9 with a series of double-stranded nucleic acid substrates having different sequences.

[0088] In some embodiments, the method includes screening, testing, or comparing several reaction conditions in order to identify preferred or optimal reaction conditions for Cas9. In these embodiments, the method comprises contacting a series of reaction mixtures comprising different buffer configurations with the same Cas9. In some embodiments, the series of reaction mixtures is also subjected to different temperature profile during the Cas9 cleavage step.

[0089] The method next comprises measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates activity of Cas9. The change in fluorescence includes a change in color (wavelength) of the fluorescent signal as well as appearance of fluorescence where fluorescence was previously not detectable. In some embodiments, the measurement is qualitative indicating the presence or absence of Cas9 activity. In other embodiments, the measurement is quantitative indicating the relative amount of Cas9 activity. In some embodiments, the change in fluorescence further includes a change in the intensity of fluorescence and the difference in fluorescence among the samples being tested.Attorney Docket Number: CBI060.30PATENT

[0090] In some embodiments the method described herein is performed with isolated nucleic acid substrates and isolated polypeptides. In other embodiments, the assay is performed with crude mixtures without substantial purification steps. In some embodiments, an isolated or purified nucleic acid substrate is added to crude isolates or emergent fractions of Cas9, for example, to rapidly assess the Cas9 production or purification process. In some embodiments, isolated or purified Cas9 polypeptides are added to crude isolates of nucleic acids, for example, minimally treated patient samples to rapidly detect the presence of an infectious agent in the patient.

[0091] In some embodiments, the invention is a double stranded nucleic acid substrate and a method of preparing the substrate.

[0092] The substrate comprises a donor fluorophore (reporter) and an acceptor fluorophore (quencher). The reporter and a quencher may be located on the same strand of the double stranded nucleic acid substrate. For example, one strand may have a thymine-linked fluorescein and an Iowa Black® quencher.

[0093] The double stranded nucleic acid substrate may be prepared by combining the two strands (the target strand and the complementary non-target strand) in a reaction mixture comprising a suitable buffer (e.g., TE). For optimal annealing, the mixture may be heated to >90°C and allowed to cool to room temperature.

[0094] In some embodiments, it may be desirable to perform the assay described herein in crude preparations of nucleic acids where exonucleases may be present. In such situations the double stranded nucleic acid substrate further comprises exonuclease protection (e.g., 1, 2, 3, 4, or about 5 phosphorothioate bonds at the 3’ ends of both strands of the substrate.

[0095] The substrate is contacted with Cas9 in a suitable buffer under suitable reaction conditions that can be obtained published studies, e.g., Gasiunas, G., et al., (2020)^4 catalogue of biochemically diverse CRISPR-Cas9 orthologs, Nature Comm. 11 :5512. In some embodiments, the reaction buffer is a commercial low-salt endonuclease buffer, e.g., NEB Buffer 1 or CutSmart® buffer (New England Biolabs, Ipswich, Mass.)

[0096] In some embodiments, a nucleoprotein complex (e.g., a ribonucleoprotein complex, RNP) including Cas9 is assembled and added to the reaction mixture. The nucleoprotein complex comprises Cas9 and the nucleic acid targeting nucleic acid (NATNA). To assemble the nucleoprotein complex, the NATNTA is incubated with Cas9 under suitable conditions e.g., 37°CAttorney Docket Number: CBI060.30PATENT for 10 minutes. NATNTA can be pretreated to allow for proper secondary structure formation, by heating (e.g., to 95 °C for 2 minutes) and allowed to slowly cool to room temperature.

[0097] Fluorescence of the reaction mixture is measured following the addition of all the reaction components including the substrate and the RNP.

[0098] In some embodiments, linear range of the assay with respect to the concentration of Cas9, and the concentration of the nucleic acid substrate is tested to determine the optimal range and sensitivity of the assay with respect to Cas9 concentration and substrate concentration.

[0099] In some embodiments, the invention is a composition for detecting activity of Cas9. The composition includes the nucleic acid substrate described herein comprising: a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer and a protospacer adjacent motif (PAM) for a Cas9; and a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9. In some embodiments, the 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’ and a NATNA capable of binding Cas9 and enabling cleavage by Cas9 and posttrimming activity of Cas9.

[0100] In some embodiments, the assay is used to validate or to optimize a genome editing protocol that utilizes Cas9. In that case, the substrate sequence is selected from a genomic locus to be edited by the Cas9. In some embodiments, the substrate comprises a sequence from the human TRAC locus. In some embodiments, the substrate comprises a sequence selected from SEQ ID NOs: 1-8. In some embodiments, the substrate comprises a sequence from the human PDCD1 locus. For illustration purposes only, the nucleic acid strands of the substrate are arbitrarily designated “top” and “bottom” to indicate complementary strands forming a duplex. In some embodiments, the substrate comprises a sequence selected from SEQ ID NOs: 9-38. In some embodiments, both fluorophores are on the top strand (SEQ IDs: 1, 3, 5, 7, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37). In some embodiments, both fluorophores are on the bottom strand (SEQ IDs: 10, 12, 14, 16, and 18). In some embodiments, the fluorophore containing strand contained a bulge, i.e., an unpaired nucleotide, wherein the fluorophore is conjugated to the unpaired nucleotide. In some double stranded substrates, the top strand (SEQ IDs: 1, 3, 5, 25 and 29) formed a bulgeAttorney Docket Number: CBI060.30PATENT comprising the fluorophore-conjugated nucleotide (the bottom strand did not have a nucleotide to pair with the fluorophore-conjugated nucleotide). In some double stranded substrates, the bottom strand (SEQ IDs: 12 and 18) formed a bulge comprising the fluorophore-conjugated nucleotide (the top strand did not have a nucleotide to pair with the fluorophore-conjugated nucleotide).

[0101] In some embodiments, the invention is a method of measuring activity of a Cas9 endonuclease in real time. In some embodiments, the measurement is performed in a reaction mixture comprising a substrate at 100 nm, Cas9 at 50 nM, ch-acr (DNA-containing tracrRNA) at about lOOnM, and chRDNA (DNA-containing crRNA) at 1.56 nM to 100 nM. In some embodiments, the reaction is performed in a commercial low-salt endonuclease buffer (e.g., NEB Buffer 1 (New England Biolabs, Ipswich, Mass.). In some embodiments, the reaction mixture is incubated 37°C for 2 hours and fluorescence is measured continuously with the use of a fluorometer.

[0102] In some embodiments, the assay possesses a linear range (linearity) within a range of concentration of reactants. In some embodiments, linearity with respect to guide RNA (chRDNA) concentration is assessed in reaction mixtures comprising the substrate described herein at about lOOnM, Cas9 at about 50nM, or 75 nM, or 150 nM, ch-acr (DNA-containing tracrRNA) at about lOOnM, while the concentration of chRDNA (DNA-containing crRNA) is varied between 1.56 nM and 100 nM or between 4.6 nM - 75 nM. In some embodiments, linearity with respect to DNA substrate concentration in reaction mixtures comprising Cas9 at about 50nM, ch-acr (DNA-containing tracrRNA) at about lOOnM, and chRDNA (DNA-containing crRNA) at about 1.56 nM or 100 nM, while the concentration of the substrate is varied between 25 and 100 nM. In some embodiments, linearity with respect to nucleoprotein complex (NPC) concentration is assessed in reaction mixtures comprising ch-acr (DNA-containing tracrRNA) at about lOOnM, substrate described herein at about lOOnM, while the concentration of the NPC comprising Cas9 and chRDNA is varied between 6.25 and 150 nM or between 12.5 and 150 nM. In some embodiments, linearity with respect to DNA-containing tracrRNA (ch-acr) concentration is assessed in reaction mixtures comprising substrate described herein at about lOOnM, Cas9 at about 50nM, and chRDNA (DNA-containing crRNA) at about 1.56 nM or 100 nM, while the concentration of ch-acr is varied between 4.6 and 75 nM.Attorney Docket Number: CBI060.30PATENT

[0103] In some embodiments, the invention provides a method suitable for use as a convenient tool for assessing, screening, testing, or comparing several preparations of Cas9. The method further allows to assess a set of conditions for Cas9 cleavage by allowing to determine which of the conditions permits the highest level or rate of Cas9 activity. The method further allows for assessing Cas9 isolation and purification methods. Specifically, the method can be applied to compare protein isolation fractions to identify the fraction containing the isolated Cas9 protein. In such embodiments, modifications are made to ensure that all the components, e.g., the Cas9, the nucleic acid substrate and the NATNA are capable of being active and are at least partially protected from enzymatic degradation in the crude preparation. The method can be rapidly applied to nascent fractions, e.g., to monitor the Cas9 purification process. Yet further, the method may be used to screen multiple Cas9 substrates having different sequences to quickly identify the best substrate, or multiple NATNAs to quickly identify the best NATNA.

[0104] The methods and compositions disclosed herein can be used in a diagnostic assay. In some embodiments, the invention is a method of detecting the presence of a target nucleic acid in a sample, wherein the nucleic acid comprises a recognition sequence for Cas9. In some embodiments, the sample is a patient’s sample. In some embodiments, the target nucleic acid may be characteristic of a microorganism, including a virus or a bacterium. In other embodiments, the target nucleic acid may comprise a polymorphism or a sequence whose presence is related to a disease or condition to be detected in the patient.

[0105] The method involves manipulating nucleic acids from a sample. In some embodiments, the sample is derived from a subject or a patient. In some embodiments the sample may comprise a fragment of a solid tissue or a solid tumor derived from the subject or the patient, e.g., by biopsy. The sample may also comprise body fluids that may contain nucleic acids e.g., urine, sputum, serum, blood, or blood fractions, i.e., plasma, lymph, saliva, sputum, sweat, tear, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric fluid, intestinal fluid, or fecal samples). In other embodiments, the sample is a cultured sample, e.g., a tissue culture containing cells and fluids from which nucleic acids may be isolated. In some embodiments, the nucleic acids of interest present or suspected of being present in the sample come from infectious agents such as viruses, bacteria, protozoa or fungi.Attorney Docket Number: CBI060.30PATENT

[0106] In some embodiments, the diagnostic method includes a preliminary amplification (pre-amplification) procedure wherein the nucleic acid in the sample is amplified via a method selected from the polymerase chain reaction (PCR), or an isothermal amplification method selected from recombinase-polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP or RT-LAMP) and nucleic acid sequence-based amplification (NASBA) to generate an amplicon from each target nucleic acid.

[0107] In some embodiments, the Cas9 substrate, which is a probe comprising a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer and a protospacer adjacent motif (PAM) for a Cas9 and a donor fluorophore and an acceptor fhiorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9 is hybridized to a target nucleic acid or an amplicon of a target nucleic acid in a sample. In some embodiments, the probe, the target nucleic acid or the amplicon are single-stranded. In some embodiments, the probe, the target nucleic acid or the amplicon are double-stranded but are rendered single-stranded prior to being hybridized to the probe. The duplex formed by the target nucleic acid hybridized to the probe and used as the Cas9 substrate in the method disclosed herein.

[0108] The nucleic acid substrate formed by any of the alternative methods described above comprises a diagnostically relevant nucleic acid sequence to be interrogated. In some embodiments, the nucleic acid substrate is contacted by Cas9 and NATNA (e.g., as an RNP complex) targeting the sequence of interest, e.g., a sequence characteristic of a microorganism or a sequence comprising a polymorphism or a sequence whose presence is related to a disease or condition to be detected in a patient. The Cas9 performs cleavage and fluorescence is detected only if the target nucleic acid is present in the sample.

[0109] In some embodiments, the diagnostic method is multiplex, z.e., multiple target sequences are detected in the same reaction mixture. In such embodiments, multiple nucleic acid probes are added to the sample. In some embodiments, Cas9 is paired with multiple NATNAs and Cas9 performs cleavage leading to generation of a detectable signal when any of the target sequences capable of hybridizing to any of the NATNAs is present. In some embodiments, each of the different probes is labeled with a different fluorophore. In some embodiments, all or some of the different probes are labeled with the same fluorophores, e.g., one label for a set of probesAttorney Docket Number: CBI060.30PATENT hybridizing to bacterial sequences, and another label for a set of probes hybridizing to viral sequences, or one label for a set of probes hybridizing to Gram-positive bacterial sequences, and another label for a set of probes hybridizing to Gram-negative bacterial sequences. The presence of the fluorescent signal indicated the presence of the sequence of interest in the sample. In some embodiments, the assay includes the step of reporting that the sequence of interest (e.g., a sequence characteristic of a microorganism or polymorphism or a sequence whose presence is related to a disease or condition to be detected in a patient) is present in the sample.

[0110] In some embodiments, the invention is a kit for detecting activity of Cas9. The kit includes an aliquot of the nucleic acid substrate described herein. The nucleic acid substrate comprises a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer and a protospacer adjacent motif (PAM) for a Cas9; and a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9. In some embodiments, the 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’. In some embodiments, the kit further comprises one or more NATNAs capable of binding the substrate and the Cas9.[OHl] In some embodiments, the kit further comprises instructions on performing the method of testing for activity of Cas9 by the method described herein.

[0112] In some embodiments, the invention is a kit for performing a diagnostic procedure. The kit includes an aliquot of a probe capable of hybridizing to a target nucleic acid of diagnostic interest and comprising a target strand forming a hybrid with a non-target strand, wherein the non- target strand comprises a spacer and a protospacer adjacent motif (PAM) for a Cas9; and a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming (PCT) zone of the Cas9. In some embodiments, the 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’. The diagnostic kit may further comprise one or more NATNAs capable of hybridizing to one or more sequences of interest. The diagnosticAttorney Docket Number: CBI060.30PATENT kit may further comprise Cas9. In some embodiments, the kit further comprises one or more NATNAs capable of binding the substrate and the Cas9. In some embodiments, the kit further comprises instructions on performing the diagnostic assay described herein.

[0113] The method disclosed herein may be performed with a specialized apparatus. In some embodiments, the invention is an apparatus for detecting activity of Cas9 comprising one or more reaction chambers for performing enzymatic reactions and a fluorescence detector. In some embodiments, the apparatus is capable of repeated (timed) fluorescence measurements. In some embodiments, the apparatus is capable of continuous measurement of fluorescence. With repeated or continuous measurement of fluorescence, the apparatus is capable of real-time assessment of Cas9 activity.

[0114] The apparatus may further comprise means for delivering and dispensing components of the reaction mixtures described herein. The apparatus may be adapted for high- throughput screening, e.g., in multiwell plates (microwell plates). The apparatus may comprise a multiwell plate fluorescence reader or a tube fluorometer such as the ones available from Tecan, ThermoFisher Scientific (BioTek instruments), and Molecular Devices.EXAMPLES

[0115] Example 1. Substrate design

[0116] Each substrate comprised a double stranded nucleic acid having a top strand and a bottom strand (SEQ ID NOs: 1-38). The substrates contained the sequences of the human 77MC gene (SEQ ID NOs: 1-8) or the human PDCD1 gene (SEQ ID NOs: 9-38). The nucleic acid strands are arbitrarily designated “top” and “bottom” to indicate complementary strands forming a duplex.

[0117] For TRAC substrates 6, 7, 8 and 9, the top strand (SEQ IDs: 1, 3, 5, and 7) comprised a fluorophore and a quencher. In TRAC substrates 6, 7, and 8, the top strand (SEQ IDs: 1, 3, and 5) formed a bulge comprising the fluorophore-conjugated nucleotide (the bottom strand did not have a nucleotide to pair with the fluorophore-conjugated nucleotide). In the TRAC substrate 9, the bottom strand was perfectly complementary to the top strand.

[0118] For PDCD1 substrates 2-6, the bottom strand (SEQ IDs: 10, 12, 14, 16, and 18) comprised a fluorophore and a quencher. For PDCD1 substrates 7-16, the top strand (SEQ IDs: 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37) comprised a fluorophore and a quencher. InAttorney Docket Number: CBI060.30PATENTPDCD 1 substrates 3 and 6, the bottom strand (SEQ IDs: 12 and 18) formed a bulge comprising the fluorophore-conjugated nucleotide. In PDCD1 substrates 10 and 12, the top strand (SEQ IDs: 25 and 29) formed a bulge comprising the fluorophore-conjugated nucleotide. In all the other PDCD1 substrates the two strands were perfectly complementary.

[0119] Example 2. Measuring Cas9 activity with DNA substrates.

[0120] This experiment was performed with Substrates 6 and 7. The reactions contained fixed concentrations of substrates at 100 nM, Cas9 at 50 nM, ch-acr (DNA-containing tracrRNA) at 100 nM, and chRDNA (DNA-containing crRNA) at 100 nM. The reaction was performed in NEB Buffer 1 (New England Biolabs, Ipswich, Mass.). The reaction was incubated at 37°C for 12 hours. Fluorescence was measured continuously every 10 minutes on a Spectramax. Results are shown in FIGURE 3.

[0121] Example 3. Assay linearity with respect to guide RNA (chRDNA) concentration.

[0122] This experiment was performed with Substrate 7. The reactions contained fixed concentrations of substrate (Substrate 7) at 100 nM, Cas9 at 50nM, ch-acr (DNA-containing tracrRNA) at 100 nM. The concentration of chRDNA (DNA-containing crRNA) was varied between 1.56 nM and 100 nM. The reaction was performed in 20 mM HEPES:KOH pH 7.5, 100 mM KC1, 5 mM MgC12, 5% glycerol. The reaction was incubated at 37° C for 2 hours. Fluorescence was measured on a Spectramax i3x (Molecular Devices, San Jose, Cal.). The signal in RFUs was converted into pmoles based on a fluorescein standard curve, and the TO value was subtracted from all the time points to give a T0=0 pM. Then the signal from the zero chRDNA conditions was subtracted from the signal for the other conditions. The slope was determined from the linear portion of the plot for each condition. Due to varying lag times and saturation points, this occasionally resulted in the rate being extrapolated from different time periods for different condition. Results are shown in FIGURE 4.

[0123] Example 4. Assay linearity with respect to DNA substrate concentration.

[0124] This experiment was performed essentially as described in Example 3 with Substrate 7, except concentration of chRDNA and ch-acr were fixed 300 nM, and Cas9 was at 150 nM. The concentration of the substrate was varied between 25 and 100 nM. The assay was monitored for 2 hours, and the slopes were calculated between in the linear region between 6 and 18 minutes. Results are shown in FIGURE 5.Attorney Docket Number: CBI060.30PATENT

[0125] Example 5. Assay linearity with respect to nucleoprotein complex (NPC) concentration.

[0126] This experiment was performed essentially as described in Example 3 with Substrate 8 except the concentration of the NPC comprising Cas9 and chRDNA was varied between 6.25 and 150 nM or between 12.5 and 150 nM, with the chRDNA and ch-acr always in in 2-fold excess relative to the Cas9. The signal in RFUs was converted into pmoles as in Example 3. The fluorescence signal was monitored every 1 minute for 90 minutes. Results are shown in FIGURE 6

[0127] Example 6. Assay linearity with respect to guide RNA (chRDNA) concentration

[0128] This experiment was performed essentially as described in Example 3 except Substrate 8 was used and the ch-acr and Cas9 concentrations were fixed at 150 nM and 75 nM respectively, and the chRDNA concentration varied in the range 1.56 - 150 nM. The fluorescence signal was monitored every 1 minute for 90 minutes. Results are shown in FIGURE 7.

[0129] Example 7. Assay linearity with respect to DNA-containing tracrRNA (ch-acr) concentration.

[0130] This experiment was performed essentially as described in Example 6 withSubstrate 8 except the chRDNA and Cas9 concentrations were fixed at 150 nM and 75 nM respectively, and the concentration of the ch-acr was varied between 2.3 and 150 nM. The fluorescence signal was monitored every 1 minute for 90 minutes. Results are shown in FIGURE 8

[0131] Example 8. Comparing two substrates

[0132] This experiment was performed essentially as described in Example 6, except that the Cas9 concentration was 75 nM, and the ch-acr and chRDNA concentrations were 150 nM. The substrates were Substrate 8 (no end protection, extra T base near the cut site on the fluorophore strand only, the fluorophore is conjugated to that extra T) and Substrate 9. The fluorescence signal was monitored every 1 minute for 90 minutes. Results are shown in FIGURE 9 (TR-S8 - Substrate 8, TR-S9 - Substrate 9).

[0133] Example 9. PDCD1 substrates

[0134] This experiment was performed essentially as described in Example 3 with Substrates 3, 4, 5 and 6, except that the concentrations of Cas9 was 75 nM, and the concentrationsAttorney Docket Number: CBI060.30PATENT of the chRDNA and ch-acr were 150 nM. The fluorescence signal was monitored every 1 minute for 90 minutes. Results are shown in FIGURE 10.

[0135] While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

Claims

Attorney Docket Number: CBI060.30PATENTWhat is claimed is:

1. A nucleic acid substrate for detecting activity of a CRISPR Cas9 endonuclease, the substrate comprising:(i) a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer and a protospacer adjacent motif (PAM) for a Cas9 endonuclease; and(ii) a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming zone of the Cas9 endonuclease.

2. The substrate of claim 1, wherein at least one of the donor fluorophore and the acceptor fluorophore is on the non-target strand.

3. The substrate of claim 1, further comprising a structure inhibiting cleavage of the substrate by an exonuclease selected from a hairpin, a strand overhang, and a nucleic acid modification.

4. The substrate of claim 3, wherein the nucleic acid modification comprises one or more phosphorothioate linkages.

5. A composition for detecting activity of a CRISPR Cas9 endonuclease comprising the nucleic acid substrate of claim 1, a nucleic acid targeting nucleic acid (NATNA), and a Cas9 endonuclease.

6. The composition of claim 5, wherein the NATNA is selected from a single guide and a dual guide.

7. The composition of claim 5, wherein the NATNA comprises DNA and RNA nucleotides.

8. The composition of claim 5 wherein the Cas9 endonuclease is a mutant or engineered variant.

9. A method for detecting activity of a CRISPR Cas9 endonuclease comprising:(a) contacting a Cas9 endonuclease with a nucleic acid targeting nucleic acid (NATNA) and a reaction mixture comprising a nucleic acid substrate comprising:Attorney Docket Number: CBI060.30PATENT(i) a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence for the Cas9 endonuclease; and(ii) a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming zone of the Cas9 endonuclease; and(b) measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates activity of the Cas9 endonuclease.

10. The method of claim 9, wherein the NATNA is selected from a single guide and a dual guide.

11. The method of claim 9, wherein the NATNA comprises DNA and RNA nucleotides.

12. A kit for detecting activity of a CRISPR Cas9 endonuclease comprising a nucleic acid targeting nucleic acid (NATNA) and a nucleic acid substrate comprising:(i) a target strand forming a hybrid with a non-target strand, wherein the non-target strand comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence for a Cas9 endonuclease; and(ii) a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair, wherein at least one of the donor fluorophore and the acceptor fluorophore is situated within the post-cleavage trimming zone of the Cas9 endonuclease.

13. The kit of claim 12, wherein the NATNA is selected from a single guide and a dual guide.

14. The kit of claim 12, wherein the NATNA comprises DNA and RNA nucleotides.

15. The kit of claim 12, wherein the substrate comprises a structure inhibiting cleavage of the substrate by an exonuclease selected from a hairpin, a strand overhang, and a nucleic acid modification.

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