Method, system and kit for the detection of PAM-free polynucleotides

Abstract

A CRISPR / Cas method for the detection of PAM-less polynucleotides is described herein. Also described herein are systems and kits for the detection of the PAM-less polynucleotides. The methods, systems and kits described herein are suitable for the detection of single or two-point mutations in target polynucleotides, miRNA and virus-associated polynucleotides.

Description

METHOD, SYSTEM AND KIT FOR THE DETECTION OF PAM-FREE POLYNUCLEOTIDESRELATED APPLICATION

[0001] The present application claims priority under applicable law to United States provisional application No. 63 / 737,199 filed on December 20, 2024, the content of which is incorporated herein by reference in its entirety and for all purposes.TECHNICAL FIELD

[0002] The present technology relates to CRISPR / Cas complex-based methods, systems and kits for the detection of protospacer adjacent motif (PAM)-free polynucleotides.BACKGROUND

[0003] CRISPR-Cas 12a (Cpfl), a class 2, type V endonuclease, is an RNA-guided nuclease that performs cis- cleavage of dsDNA or ssDNA targets and indiscriminate trans cleavage of ssDNA. The programmable and specific targeting capabilities of Casl2a have made it a widely applied tool in methods used for nucleic acid detection and gene editing. These methods provide promising next-generation diagnostic approaches for low-burden clinical-grade point-of-need testing without complex instrumentation. However, to this day, the dependence CRISPR-Cas 12a’ s nucleic acid detection capabilities on the existence of a protospacer adjacent motifs (PAMs) on the target polynucleotide sequence remains a limiting factor in widening the scope of its application. Moreover, other techniques allowing the detection of RNA with CRISPR-Cas 12a rely heavily on precise salt and pH regulation which render the implementation of such techniques challenging and labor-intensive.

[0004] As such, alternative or improved CRISPR-Cas 12a methods and systems are needed to overcome or alleviate at least some of the drawback of the existing methods.SUMMARY

[0005] According to some aspects, embodiments of the technology as described herein include the following items:1. A method for detecting a target polynucleotide in a sample comprising: a) contacting the sample with: i) a Casl2a CRISPR-associated (Cas) enzyme; ii) a CRISPR RNA (crRNA) comprising a guide sequence, the guide sequence having a protospacer adjacent motif (PAM)-proximal portion and a PAM -distal portion, the PAM- distal portion being configured to hybridize with the target polynucleotide;iii) a PAM duplex having a PAM sequence and a target strand configured to bind to the PAM-proximal portion of the guide sequence, such that upon binding to the guide sequence, a distal end of the target strand of the PAM duplex and the target polynucleotide are spaced apart from one another by at least one nucleotide, and a trans nuclease activity of Cas 12a is activated; and iv) a reporter capable of being activated by the trans nuclease activity of Cas 12a; and b) measuring a detectable signal produced by the activated reporter, wherein said measuring provides for the detection of the target polynucleotide in the sample.2. The method of item 1, wherein the at least one nucleotide is one nucleotide.3. The method of item 1, wherein the at least one nucleotide is two nucleotides.4. The method of any one of items 1 to 3, wherein the at least one nucleotide, is an at least one mismatched nucleotide between the PAM-distal portion and the target polynucleotide.5. The method of any one of items 1 to 4, wherein the target strand of the PAM duplex binds to the the first four nucleotides of the PAM-proximal portion of the guide sequence.6. The method of item 5, wherein the target strand of the PAM duplex binds to the first four nucleotides of the PAM-proximal portion of the guide sequence.7. The method of any one of items 1 to 6, wherein the target nucleotide sequence is a PAM-free single stranded DNA, a PAM-free double-stranded DNA, or an RNA.8. The method of item 7, wherein the RNA is a miRNA.9. The method of any one of items 1 to 7, wherein the target nucleotide sequence is a virus-associated polynucleotide.10. The method of item 9, wherein the virus-associated polynucleotide is SARS-CoV-2 polynucleotide, a hepatitis C virus (HCV) polynucleotide, a human immunodeficiency virus (HIV) polynucleotide, a malaria polynucleotide, or a bluetongue virus polynucleotide.11 . The method of any one of items 1 to 10, wherein the PAM duplex is blunt ended.12. The method of any one of items 1 to 10, wherein a non-target strand of the PAM duplex is longer than the target strand.13. The method of item 12, wherein the target polynucleotide is a ssDNA or RNA, and the non-target strand of the PAM duplex is configured to bind to a portion of the target polynucleotide which is not bound to the PAM-distal portion of the guide sequence.14. The method of any one of items 1 to 13, further comprising amplifying the target polynucleotide in the sample.15. The method of item 14, wherein the amplification is by LAMP, RT-LAMP, NASBA, RPA, and EXPAR.16. The method of any one items 1 to 15, further comprising contacting the sample with a DNA ligase (e.g. T4 DNA ligase) in step a).17. A nucleic acid detection system for detecting a target polynucleotide in a sample comprising: i) a Casl2a CRISPR-associated (Cas) enzyme; ii) a CRISPR RNA (crRNA) comprising a guide sequence, the guide sequence having a protospacer adjacent motif (PAM)-proximal portion and a PAM-distal portion, the PAM-distal portion being configured to hybridize with the target polynucleotide; iii) a PAM duplex having a PAM sequence and a target strand configured to bind to the PAM-proximal portion of the guide sequence, such that upon binding to the guide sequence, a distal end of the target strand of the PAM duplex and the target polynucleotide are spaced apart from one another by at least one nucleotide, and a trans nuclease activity of Cas 12a is activated; and, iv) a reporter capable of being activated by the trans nuclease activity of Cas 12a.18. The system of item 17 wherein: a) the at least one nucleotide is as defined in any one of items 2 to 4; b) the target strand of the PAM duplex binds to the guide sequence as defined in item 5 or 6; c) the target polynucleotide is as defined in any one of items 7 to 10; d) the PAM duplex is as defined in any one of items 11 to 13; or e) any combination of a) to d).19. The system of item 17 or 18, further comprising a DNA ligase (e.g. a T4 DNA ligase).20. A kit for detecting a target polynucleotide in a sample, the kit comprising the crRNA as defined in item 1 and a PAM duplex as defined in in any one of items 1, and 12 to 14.21 . The system of item 17 to 19, or the kit of item 20 for use in detecting: a) a single or a two-point mutation in a target polynucleotide; b) a miRNA; or c) a virus-associated polynucleotide.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Various objects, features and advantages of the disclosure will be apparent from the following description of particular embodiments, as illustrated in the accompanying drawings.

[0007] FIG. 1A is a schematic illustration of a traditional Casl2a system (prior art) with Casl2a introducing a staggered dsDNA break on the target nucleic acid strand when it is activated.

[0008] FIG. IB is a schematic illustration of a system according to one embodiment of the present technology using LbCasl2a.

[0009] FIG. 1C is a graph demonstrating the activation of LbCasl2a in relation to the position of the nick location (triangle) within a target dsDNA. Complete activation is observed at positions P2 an P20, partial activation at P9 and P19, and no activation at P12. n=4 technical replicates; bars represent the arithmetic mean ± SD.

[0010] FIG.l D is a graph demonstrating the activation of LbCasl2a in relation to the position of the nick location within a target dsDNA. Complete activation is observed at positions P10 an P21, partial activation at P13 and P20, and no activation at P12 and P14. n=4 technical replicates; bars represent the arithmetic mean ± SD.

[0011] FIG. 2A is a schematic illustration of six configurations of the RAPID system tested. Type I is a configuration with continuous elongation of the target strand towards the PAM -distal region, while the non-target strand remains fixed. Type II is similar to (I), but with elongation extending into the PAM motif. Types III & IV are configurations analogous to (I) and (II), respectively, but without a nontarget strand. Type V Utilizes ssDNA with strategic single nucleotide nick placements (triangle) within the protospacer region. Type VI Features PAM containing dsDNA with single-stranded breaks on the target strand.

[0012] FIG. 2B is a heat map showing normalized fluorescence intensities resulting from Casl2a activation and trans cleavage of the reporter for the six configurations described in FIG. 2A, across 24 nick locations (Pl to P24) within the protospacer region, including the PAM. n=4 technical replicates.

[0013] FIG. 3A is a schematic illustration of a selection of single nucleotide nick positions on the target strand of PAM-containing dsDNA.

[0014] FIG. 3B is a bar graph illustrating the normalized / ram-clcavagc fluorescence intensity at the nick positions of the PAM-containing dsDNA of FIG. 3A.

[0015] FIG. 3C is aphotograph of an agarose gel electrophoresis displaying the / ram-cleaved bands of cpX174 virion circular ssDNA corresponding to the nicked positions, compared to the cpX174 virion DNA template.

[0016] FIG. 3D is a schematic illustration of a randomly selected nick positions on the non-target strand of a PAM-containing dsDNA at positions S9, SI 1, and SI 6.

[0017] FIG. 3E is a bar graph illustrating the normalized / ram-cleavage fluorescence intensity at the nick positions of FIG. 3D, indicating no significant Cas 12a activation differences.

[0018] FIG. 3F is a photograph of an agarose gel demonstrating the electrophoretic mobility of trans- cleaved cpX174 virion DNA template, which was completely cleaved at these positions, suggesting that Casl2a's tunability is not evident when the non-target strand is nicked.

[0019] FIG. 3G is a bar graph depicting trans-cleavage fluorescence intensity across all considered nick positions on the target strand of dsDNA (Pl to P24).

[0020] FIG. 3H is a heatmap showing the activation pattern of Cas 12a at selected single nucleotide nick positions across the six system types of FIG. 2A with a different gRNA (SEQ ID NO: : 58)

[0021] FIG. 4A is a schematic representation of various PAM-free detection methods across various nucleic acid types: short and long single-stranded nucleic acids (configurations Al and All), and short (blunt- ended) (configuration III) and long double-stranded DNA (dsDNA) (configuration IV). Configuration Al and All illustrate PAM duplexes with sticky ends, while configurations III and IV illustrate blunt- ended PAM-duplexes.

[0022] FIG. 4B is a bar graph demonstrating the performance of RAPID in detecting various lengths of target ssDNA (28-nt, 89-nt, 139-nt) compared to PAM-containing dsDNA (PAM) and a non-target control (NTC).

[0023] FIG. 4C is a bar graph demonstrating the performance of RAPID for target RNA lengths of 28-nt, 65- nt, and 116-nt, demonstrating better performance with shorter RNA. AsCasl2a exhibits higher background noise with NTC compared to LbCasl2a.

[0024] FIG. 4D is a bar graph demonstrating fold changes in fluorescence intensity based on the detection on a dsDNA with a universal (blunt-ended) PAM-duplex (configuration III of FIG. 4A) at various concentrations (from 0 to 1000 nM) at 120 minutes. The dark grey bar represents the benchmark using PAM-containing dsDNA. Experiments were conducted with LbCasl2a and blunt-ended dsDNA target at a final concentration of 10 nM.

[0025] FIG. 4E is a bar graph demonstrating fold change in fluorescence intensity based on universal (blunt- ended) PAM-duplex concentrations (from 0 to 1000 nM) at 120 minutes in configuration IV of FIG. 4A, compared to the signal from PAM-containing dsDNA (dark grey bar). Experiments were conducted with LbCasl2a; PAM free dsDNA at a final concentration of 10 nM.

[0026] FIG. 4F is a bar graph demonstrating the performance of RAPID for the detection of PAM-free blunt- ended target dsDNA (52-bp) and longer PAM-free dsDNA (139-bp), compared to PAM-containing dsDNA and NTC controls.

[0027] FIG. 4G is a kinetic plot comparing fluorescence intensities from blunt-ended PAM-free dsDNA (52- bp) and PAM-free dsDNA (139-bp) to PAM-containing dsDNA with AsCasl2a, indicating similar fluorescence intensities. NTC (negative control) shows the intrinsic high background signal of AsCasl2a. All dsDNAs were at a final concentration of 50 nM.

[0028] FIG. 4H is a kinetic plot comparing fluorescence intensities from blunt-ended PAM-free dsDNA (52- bp) and PAM-free dsDNA (139-bp) to PAM-containing dsDNA with LbCasl2a. All dsDNAs were tested at a concentration of 50 nM.

[0029] FIG. 5A left panel is a schematic illustration of the position of single point mutations on ssDNA relative to the trans PAM duplex tested, with nucleotide 1 being proximal to the trans PAM and nucleotide 16 being distal; the right panel is a bar graph showing the trans cleavage rate at each position. Bar numbering corresponds to mutation sites. n=3 technical replicates; bars represent the arithmetic mean ± SD. Final ssDNA concentration is 2 pM, Trans-cleavage rate is analysed within 2 h. Experiments were compared to the wild-type sequence and an NTC.

[0030] FIG 5B left panel is a schematic illustration of the position of the two-base mismatch in the ssDNA relative to the trans PAM duplex tested, with nucleotide 1 being proximal to the trans PAM and nucleotide 16 being distal; the right panel is a bar graph showing the trans cleavage rate at each position. Bar numbering corresponds to mutation sites. n=3 technical replicates; bars represent the arithmetic mean ± SD. Final DNA concentration is 2 pM. Trans-cleavage rate is analysed within 2 h. Experiments were compared to the wild-type sequence and an NTC.

[0031] FIG. 5C left panel is a schematic illustration of the position of single-point mutations in a 16-nt region within the protospacer of miRNA-21 relative to the trans PAM duplex with nucleotide 1 being proximal to the trans PAM and nucleotide 16 being distal, with the remainder binding to the PAM duplex DNA. Results are expressed as trans-cleavage rate per min. Bar numbering corresponds to mutation sites. n=3 technical replicates; bars represent the arithmetic mean ± SD. Final miRNA is 50 nM. Trans-cleavage rate is analysed within 2 h. Experiments were compared to the wild-type sequence and an NTC.

[0032] FIG. 6A illustrates the results of FIG. 5A after 60 minutes of reaction time (instead of 2h), the results are expressed in relative fluorescence unit (RFU).

[0033] FIG. 6B illustrates the results of FIG. 5B after 60 minutes of reaction time (instead of 2h), the results are expressed in relative fluorescence unit (RFU).

[0034] FIG. 6C illustrates the results of FIG. 5C after 60 minutes of reaction time (instead of 2h), the results are expressed in relative fluorescence unit (RFU).

[0035] FIG. 7A is a bar graph showing the fluorescence intensity of different lengths of RNA (29-nt, 65-nt, and 116-nt) with RAPID in the presence of various concentrations of MgCk

[0036] FIG. 7B is a schematic illustration of the detection miRNA by the methods of the present technology.

[0037] FIG. 7C is a heat map displaying the optimization of gRNA, AsCasl2a, and PAM duplex concentrations for the method of FIG. 7B. Incubation time was for 1 h. n=3 technical replicates.

[0038] FIG. 7D is a bar graph showing the fluorescent intensity resulting of the methods of FIG. 7B in the presence of various concentrations of PEG8000 (ranging from 0 to 30%). The (+) and (-) signs represent reactions with and without the miRNA target.

[0039] FIG. 7E is a kinetic plot of miRNA-21 detection using RAPID, analyzed over a 120-minute period.

[0040] FIG. 7F is a bar graph showing the limit of detection for miRNA-21 using RAPID, achieving a sensitivity down to 160 pM, with statistical significance compared to the NTC. Incubation time was 1 h. n=3 technical replicates; bars represent the arithmetic mean ± SD.

[0041] FIG. 7G is a graph showing the limit of detection of miRNA-21 calculated from 3o / S, where o is the standard deviation of the background signal and S is the slope of the fluorescence of target concentration line.

[0042] FIG. 7H is a heat map displaying orthogonality testing of RAPID for three miRNAs: miRNA-21, miRNA-320, and miRNA-210. Incubation times were for 1 h. n=3 technical replicates. NTC refers to no template control.

[0043] FIG. 71 is a bar graph from orthogonality testing of RAPID for miRNA-21 , miRNA-320, and miRNA- 210. The data, plotted after 2 hours, shows 100% orthogonality, confirming the system's specificity.

[0044] FIG. 7J is a bar graph showing the stability of the method of FIG. 7B when the RAPID components are freeze-dried for various time periods. Incubation time was for 1 h. n=3 technical replicates; bars represent the arithmetic mean ± SD. The (+) and (-) signs represent reactions with and without the miRNA target.

[0045] FIG. 7K is a kinetic plot for freeze-drying stability of miRNA-21 over 7 days.

[0046] FIG. 8A a schematic illustration of the workflow from the collection to the detection of target RNA in patient samples using RAPID system coupled with LAMP.

[0047] FIG. 8B is a schematic illustration of the interaction of RAPID with a loop region of a LAMP- generated dumbbell structure.

[0048] FIG. 8C is a bar graph showing RAPID detection of ssDNA strands at a 1 nM concentration that mimic the loops of SARS-CoV-2 LAMP dumbbells.

[0049] FIG. 8D is a graph showing the detection of SARS-CoV-2 viral RNA in samples with 100 copies / pL (positive) and control (negative, NTC) samples, showing that RNA detection with RAPID and LAMP can be completed within 15 minutes.

[0050] FIG. 8E is a bar graph showing the volume of LAMP reactions spiked into the RAPID reaction mix.

[0051] FIG. 8F is a bar graph showing the ultra-sensitivity of RAPID with LAMP, detecting as low as 0.1 copies / pL within 30 minutes.

[0052] FIG. 8G is a graph showing the fluorescence signal detected for varying concentrations of a SARS- CoV-2 synthetic target over time with RAPID-LAMP, detecting down to 0.1 copies / pL within 5 minutes.

[0053] FIG. 8H is a bar graph showing the detection of various SARS-CoV-2 variants using RAPID coupled with LAMP, all within 30 minutes.

[0054] FIG. 81 is a kinetic plot showing the fluorescence signal detected for various SARS-CoV-2 variants, including Wuhan virus, Alpha VOC, Beta VOC, Gamma VOC, Delta VOC, and Omicron VOC with RAPID-LAMP.

[0055] FIG. 8J is a bar graph showing RAPID's selectivity against RNAs from other viral targets.

[0056] FIG. 8K is a kinetic plot of RAPID-LAMP’s selectivity against various viral RNAs, including H1N1, H7N9, 229E, NL63, OC43, MERS-CoV, SARS-CoV-1, and SARS-CoV-2, where the system is shown to have 100% selectivity.

[0057] FIG. 9A is a scatter plot showing the results from 25 patient samples tested with RAPID-LAMP, with 14 negative and 11 positive for SARS-CoV-2 RNA.

[0058] FIG. 9B is a scatter plot showing the Ct values for RNase P in the 25 patient samples of FIG. 9A, distinguishing between positive and negative samples.

[0059] FIG. 9C is a bar graph showing fluorescence intensity of the freeze-dried RAPID-LAMP assay up to 7 days. Results after 2 hours of incubation at 37 °C (Methods 11-12). n=3 technical replicates; bars represent the arithmetic mean ± SD. (+) and (-) represent SARS-COV-2 positive and NTC samples respectively.

[0060] FIG. 9D is a graph showing the fluorescence signal of the stability assay of FIG. 9C.

[0061] FIG. 9E is a bar graph showing fluorescence intensity of the freeze-dried RAPID-LAMP assay after 2 hours, demonstrating robust stability of the developed tool.

[0062] FIG. 10A (left )is a schematic illustration of nicked DNA repair using T4 DNA ligase at position P12. The 5' end of the nicked DNA is phosphorylated, enabling repair in the presence of ligase. (+) indicates the addition of ligase enzyme, (-) denotes its absence. (Right) is a bar graph illustrating the RFU.min in each group which represents the area under the curve calculated from kinetic data within 60 minutes. Data represent mean ± standard deviation from n = 3 technical replicates.

[0063] FIG. 10B is a heat map illustrating the ligation activity of nicked DNA at various positions.

[0064] FIG. 10C (left) is a schematic illustration of miRNA ligation to a DNA substrate, forming a chimeric DNA-RNA configuration in a single reaction. (+) indicates the presence of miRNA template, and (-) denotes its absence. (Right) is a bar graph illustrating the fluorescence in each group over time. Data represent mean ± standard deviation from n = 3 technical replicates.

[0065] FIG. 10D is a bar graph comparing ligation efficiency of miRNA at various positions (P5, P8, P9, P12, and NTC). (+) and (-) denote the presence or absence of ligase enzyme, respectively.

[0066] FIG. 10E Kinetic graph illustrating ligation activity at position P8. Symbols (+), (-), and NTC correspond to the conditions described in FIG. 10D. Data represent mean ± standard deviation from n = 3 technical replicates.SEQUENCE LISTING

[0067] This application contains a Sequence Listing in computer readable form created on December 18, 2025. The computer readable form is incorporated herein by reference.*The region in bold is part of the protospacer region that binds to the gRNA **The sequences underligned represent the mismatches.***IVT denotes in vitro transcription* Sequence in bold is miRNA-21 when it is transcribed ** Underlined sequence is the domain that bind to gRNA ***Letters in lowercase denote point mutations**IDT_Duplexing means that the duplexing was done by IDT and shipped dry to the inventors*PCR_IVT means that PCR was performed to amplify the ssDNA substrate before performing in vitro reaction**IDT_Duplexing means that the duplexing was done by IDT and shipped dry to the inventors ***U.S. CDC recommended protocol fortesting patient samples.DETAILED DESCRIPTION

[0068] The following detailed description and examples are illustrative and should not be interpreted as further limiting the scope of the invention. On the contrary, it is intended to cover all alternatives, modifications and equivalents that can be included as defined by the present description. The objects, advantages and other features of the methods, systems and kits will be more apparent and better understood upon reading the following non-restrictive description and references made to the accompanying drawings.

[0069] All technical and scientific terms and expressions used herein have the same definitions as those commonly understood by the person skilled in the art when relating to the present technology. Thedefinition of some terms and expressions used herein is nevertheless provided below for clarity purposes.

[0070] Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0071] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

[0072] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0073] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

[0074] As used herein, the terms " target polynucleotide " and “target sequence”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The methods of the present technology thus determine whether the target polynucleotide is present in a sample and if so at which level or concentration.

[0075] As used herein, the term "sample" encompasses a clinical sample, including for example, a tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, aspirate, and the like. The “sample” as used herein may include normal and / or diseased cells or may be suspected of comprising disease cells. The definition includes biological fluids derived therefrom from both normal and diseased cells isolated from the sample, including for example, a cell lysate, culture supernatant (i.e., medium) or other cell extract comprising polynucleotides and / or polypeptides.

[0076] As used herein, “trans nuclease activity” or “trans cleavage” refers to the non-specific cleavage of nontarget polynucleotides, such as single-stranded DNAs (ssDNAs), which in some embodiments may be comprised in a reporter (as defined herein), by Casl2a.

[0077] As used herein the term “CRISPR RNA” or “CrRNA” refers to an RNA sequence having a constant or scaffold region at its 5' end interacting with the Cas 12a enzyme, and a guide or spacer region / sequence (also referred to as the target binding region, the complementary region, target region, or variable region) at its 3' end typically having a sequence that binds or hybridized with the target polynucleotide. The scaffold portion provides a scaffold for the Cas 12a enzyme that will traditionally cut the target polynucleotide near a PAM sequence. Thus, the portion of the crRNA guide sequence that is closer to the 5' end and scaffold portion and thus nearer to where the PAM region of a target polynucleotide would be is referred to herein as the "PAM-proximal portion" of the guide sequence, and the portion of the guide sequence further from the scaffold region and closer to the 3' end of the guide sequence is referred to herein as the "PAM-distal portion" of the guide sequence.

[0078] Broadly, the present disclosure provides methods, systems, and kits, for the detection of target polynucleotides in sample. The technology described herein stems from the discovery that introducing a nick within a target sequence modulates the trans activity of the Cas 12a enzyme in a positiondependent manner, resulting in a tunable trans nuclease activity which includes muted, semi-muted, and complete activation. Leveraging these findings, the inventors have developed a PAM-independent nucleic acid detection method that primes Cas 12a for activation using a separate PAM-duplex, independent of the target sequence, that is provided as part of the detection system, which thus allows the detection of target sequences (or target polynucleotides) that do not naturally possess the PAM sequence necessary for the binding and activation of CRISPR-Cas 12a. As such the methods of the present technology allow for the sensitive and specific detection of PAM-free dsDNA, PAM- free ssDNA and PAM-free RNA which has not been possible with conventional Cas 12 technologies. The method developed, named RNA / DNA Affinity Precision Innovative Diagnostics (RAPID), as shown below, in addition to allowing for the detection of PAM-free polynucleotides, allows for effective discrimination of single point mutations in ssDNA and RNA substrates, a challenge that is faced for traditional Casl2 and Casl3 systems. RAPID was then combined with RT-LAMP to create a SARS-CoV-2 diagnostic, which was validated with clinical samples, finding sensitive detection of about 1 aM and 100% concordance with the gold standard RT-qPCR, with Ct values of < 33 (95% confidence interval, 84-100%).

[0079] Accordingly, in a first aspect the present technology relates to methods for detecting a target polynucleotide in a sample. The method comprises contacting the sample with a Cas 12a CRISPR- associated (Cas) enzyme, a CRIPR RNA(crRNA) as described herein, a PAM duplex as described herein, a reporter capable of being activated by the trans nuclease activity of Cas 12a; and measuring adetectable signal produced by the activated reporter, wherein said measuring provides for the detection of the target polynucleotide in the sample. Specifically, the methods of the present technology allows for the Casl2a enzyme to detect both DNA and RNA and eliminates its dependency on PAM recognition for target detection by the addition of separate PAM duplexes specifically designed to optimize the trans nuclease activity of the Casl2a enzyme as will be discussed further below. The trans or collateral nuclease / cleavage activity of the Casl2a enzyme is generally known to be activated following cis-cleavage of dsDNAs, where the Casl2 enzyme becomes non-specifically activated and can subsequently cleave non-target single-stranded DNAs (ssDNAs), such as those present in reporters. Although traditional Casl2a systems are capable of detecting ssDNA as target sequences, the transcleavage activity induced by ssDNA target sequences is known to be less efficient than that induced following activation dsDNA targets, thereby hindering sensitive ssDNA target detection. It has been reported that ssDNA as a target catalyses trans-cleavage of Casl2a at a rate of about 3 turnovers per second and has a catalytic efficiency of 5 x 106 s -IM-1 , while dsDNA catalyses trans-cleavage at a rate of about 17 turnovers per second with a catalytic efficiency of 1.7 x 107 s -IM-1 [1], Moreover, traditional Casl2a systems cannot detect RNA, an important biomarker in diagnostics, without salt or pH adjustment, which is a limitation for existing methods using Casl2a [2], The methods of the present technology allows for the detection PAM-free dsDNA, ssDNA, and RNA by optimizing / enhancing the trans cleavage activity of the Casl2a enzyme, thereby facilitating the sensitive, detection of a broad range of nucleic acid targets, including miRNA and virus-associated polynucleotides, such as those from SARS-CoV-2, hepatitis C virus (HCV), human immunodeficiency virus (HIV), malaria polynucleotide, or bluetongue virus and the like, without salt or pH adjustment.

[0080] In some embodiments, the Casl2a enzyme can be selected from Casl2a enzymes such as, but not limited to ArCasl2a, AsCasl2a, BfCasl2a, BoCasl2a, BsCasl2a, CMaCasl2a, CmtCasl2a, ErCasl2a, FnCasl2a, HkCasl2a, LbCasl2a, Lb2Casl2a, Lb5Casl2a, MbCasl2a, Mb2Casl2a, Mb3Casl2a, MiCasl2a, Pb2Casl2a, PcCasl2a, PdCasl2a, PrCasl2a, PxCasl2a, and TsCasl2a. In some embodiments the Casl2a enzyme may be LbCasl2a, ErCasl2a, and / or AsCasl2a. Variants of these Casl2a enzymes with similar or enhanced endonuclease activity or trans activity are known in the art and may also be used. In some embodiments, the concentration of the Casl2a enzyme in the reaction mixture comprising the sample with the Casl2a enzyme, the crRNA, PAM duplex, and the reporter is between about 20nM and about 120 nM. In one embodiment the concentration of the Cas 12a enzyme is about 90nM.

[0081] The crRNA used in the present technology comprises a guide sequence comprising a PAM-proximal region and a PAM-distal portion as defined herein, which is capable of guiding Cas 12a binding to the target polynucleotide. The PAM-distal portion of the guide sequence is specifically designed or configured to bind to or hybridize with the target polynucleotide. As used herein the terms “hybridize”or “bind” refers to the PAM-distal portion having sufficient complementarity with the target polynucleotide to hybridize with the target sequence and direct sequence -specific binding of the CRISPR complex to the target sequence. The degree of complementarity between the PAM-distal portion of the guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, may be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP, and Maq.

[0082] The crRNA can be about or more than about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 90, 100, 110, 112, 115, 120, 130, 140, or more nucleotides in length.

[0083] In some embodiments, the crRNA may include one or more miRNA target sequences coupled to the 3' end of the cRNA. In other embodiment, the crRNA may include one or more MS2 RNA aptamers incorporated within the portion of crRNA that is not the guide sequence.

[0084] In other embodiments, the PAM-distal portion of the guide sequence of the crRNA may be less than about 150, 125, 75, 50, 45, 40, 35, 30, 25, 20, 15, 10 nucleotides in length. In some embodiments, the PAM- distal portion of the guide sequence hybridizing with the target polynucleotide is between about 10 to about 20 nucleotides. In one embodiment, the PAM-distal portion of the guide sequence hybridizing with the target polynucleotide is about 16 nucleotides in length.

[0085] In some embodiments, the concentration of the crRNA used in the reaction mixture is between about 30nM and about 120nM. In one embodiment, the concentration of the cRNA used in the reaction mixture is about 90nM.

[0086] The PAM duplex used in the present technology is a double stranded polynucleotide sequence comprising a PAM sequence and a target strand (top strand) configured to bind to the PAM-proximal portion of the guide sequence of the crRNA. The PAM sequence may be any PAM sequence known in the art which is recognized by the Cas 12a enzyme, including but not limited to, 5’-TH V-3’ (where V is A, C, or G). In certain embodiments, the PAM duplex may be blunt-ended (also referred to as a universal PAM, for example: FIG. 4A, configurations III and IV). The blunt-ended PAM duplex has the advantage of being versatile and suited for the detection of PAM-free dsDNA, ssDNA, and RNA as will be seen in the example below. It will be understood that in such embodiments the target and non-target strands of the PAM duplex have the same length. In such embodiment, the PAM duplex may be 10 nucleotides in length. In other embodiments, the PAM duplex may have sticky ends (i.e., for example, FIG. 4 A, configurations I and II). It will be understood that in such embodiments the non-target strand of the PAM duplex will be longer than the target strand. In such embodiments the non-target strand of the PAM duplex may be 46 nucleotides inlength and the target strand may be 18 nucleotides. In some embodiments, the non-target strand of the PAM duplex may be configured to bind or hybridize with a portion of the target polynucleotide, particularly single stranded DNA targets and RNA targets, which is not bound to the PAM-distal portion of the guide RNA.

[0087] The target strand of the PAM duplex is designed and configured to hybridize or bind to the PAM- proximal portion of the guide sequence of the crRNA and thus provide the PAM necessary for the activation of the CRISPR / Casl2a complex. This overcomes the limitations of existing CRISPR / Casl2a systems and methods, which are only able to target and / or detect PAM containing target dsDNA sequence and extends the capability of the CRISPR / Casl2a complex to detect PAM- free dsDNA, ssDNA and RNA targets. More specifically, the target sequence of the PAM duplex is designed such that when the target polynucleotide in the sample and target strand of the PAM duplex are bound to the PAM-distal portion and the PAM-proximal portion of the guide sequence respectively, a distal end of the target strand of the PAM duplex and the target polynucleotide are spaced apart from one another by at least one nucleotide. As discussed above and demonstrated in the examples below, by introducing this gap (or nick) in between the distal end of the target strand and the target polynucleotide sequence, the trans nuclease activity of Casl2 is optimized which increases the sensitivity and specificity of the CRISPR / Casl2a complex to target and detect PAM-free dsDNA, ssDNA, and RNA.

[0088] In some embodiments, the at least one nucleotide is one nucleotide (FIG. 2 A, configuration VI). In some embodiments, the target strand of the PAM duplex may bind the PAM-proximal portion of the guide sequence of the crRNA equivalent to P5-P24 of configuration VI of FIG. 2B. In an embodiment of interest, the target strand of the PAM duplex binds to the first four nucleotides of the PAM-proximal portion of the guide sequence of the crRNA (equivalent to P8, FIG. 2B, configuration VI).

[0089] In some embodiment, the space between the distal end of the target strand of the PAM duplex and the target polynucleotide on the guide sequence of the crRNA is provided by at least one mismatched nucleotide between the PAM-distal portion and the target polynucleotide. In such embodiments, the at least one nucleotide may be one nucleotide, thus allowing the detection of single point mutations in the target sequence. In other embodiments, the at least one nucleotide, may be two nucleotides, thus allowing the detection of double point mutations on the target sequence. As will be seen in the examples, this has allowed the detection of single and double point mutations in both ssDNA and miRNA targets by the methods of the present technology, which renders the methods suitable for the detection of viral variants, the presence of drug resistance, or the detection of oncogenic mutations, among many other applications.

[0090] In some embodiments, the concentration of the PAM duplex used in the reaction mixture is between about 1 OnM and about 120 nM. In one embodiment, the concentration of the PAM duplex in the reaction mixture is about 20nM.

[0091] The reporter may be any polynucleotide-based molecule that can be activated by the trans activity of the activated Casl2a enzyme to produce a detectable signal or a detectable molecule. A detectable signal may be any signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art. In some embodiments, the reporter may comprise an oligonucleotide element linked to a fluorophore at a first end, and linked to quencher at a second end, which quenches the fluorophore. Accordingly, the oligonucleotide element is designed so that the fluorophore and quencher are in sufficient proximity for contact quenching to occur and that when cleaved by the trans activity of the Cas 12a enzyme upon its binding and activation to the target, polynucleotide target the quencher is separated from the fluorophore and a signal is generated by the free fluorophore, thereby signaling the presence of the target polynucleotide.

[0092] Fluorophores and their cognate quenchers are known in the art and can be selected for this purpose by one having ordinary skill in the art. For example, the fluorophore may be selected from the group consisting of FITC, HEX and FAM, and the quencher may be selected from the group consisting of BHQ1, BHQ2, MGBNFQ, and 3 lABkFQ. In one embodiment, the fluorophore is 56-FAM and the quencher is 31 ABkFQ. In other embodiments, the reporter may further comprise biotin.

[0093] A detectable molecule may be any molecule that can be detected by methods known in the art. In one embodiment, the detectable molecule is one member of a binding pair and can be detected by binding to another member of the binding pair. Examples of binding pairs include, but are not limited to, antibodyantigen pairs, enzyme-substrate pairs, receptor-ligand pairs, and streptavidin-biotin.

[0094] In some embodiments, the oligonucleotide element is a ssDNA, since Cas 12a trans cleavage activity is known to preferentially cut ssDNA. Since Cas 12 enzymes preferentially cleave DNA with an A / T rich sequence, in embodiments the oligonucleotide element of the probe includes at least 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% of A and / or T. In embodiments the oligonucleotide element of the probe is a ssDNA and is about 80% of A and / or T. In some embodiments the oligonucleotide element is TA-rich or TA-only, and is about 2-10 nucleotides, preferably 6 nucleotides in length. In one embodiment, the ssDNA of the oligonucleotide element of the probe consists of A and / or T. In other embodiments, the oligonucleotide element is TTATTT.

[0095] In some embodiments, two or more types of reporters might be provided, one configured to be cleaved by an activated Cas 12a complex as detailed above to produce a first detectable signal, and a second type of reporter configured to be cleaved by a different Cas enzyme (e.g., a Cas 13b enzyme) to produce a second detectable signal, where the first and second detectable signals are distinguishable.

[0096] In some embodiments, the concentration of the reporter in the reaction mixture may be between about 50 nM and about 1 mM. In one embodiment, the concentration of the reporter is 125 nM. In another embodiment, the concentration of the reporter may be 500 nM.

[0097] In other embodiments, the method further comprises contacting the sample with the Casl2a CRISPR- associated (Cas) enzyme, the CRIPR RNA(crRNA), the PAM duplex, and the reporter (the reaction mixture) in the presence of a Magnesium salt. In embodiment, the magnesium salt is Magnesium Chloride (MgCE). In some embodiments, the concentration of the MgCE may be between about lOOnM to about 63 mM in the reaction mixture. In one embodiment, the concentration of the MgCE in the reaction mixture is 31mM. As exemplified in FIG. 7A, the presence of the MgCE enhances the fluorescence intensity and sensitivity of the methods of the present technology. In other embodiments, a crowding agent may be added alternatively or in addition to the MgCE to the reaction mixture. In some embodiments, the crowding agent may be PEG-8000. The concentration of the PEG-8000 may be between about 1.0 % w / w to about 20.0 % w / w of the reaction mixture. In one embodiment, the concentration of the PEG-8000 in the reaction mixture is about 6.0 %. As seen in FIG. 7D the presence of the PEG also increases the fluorescence intensity and sensitivity of the methods of the present technology.

[0098] In other embodiments, the method disclosed herein may further comprise amplifying the target polynucleotide in the sample, prior to or coupled with contacting the sample with the Cas 12a CRISPR- associated (Cas) enzyme, the CRISPR RNA (crRNA), the PAM duplex, and the reporter. Although, as will be seen in the examples below, the sensitivity achieved by the methods of the present technology to detect target polynucleotides, such as miRNA, were in the pico molar range, addition of an amplification step in the methods of the present technology reduced the sensitivity of the detection to about 1 aM which parallels the RT-PCR gold standard method used for the detection of SARS-CoV-2. In some embodiments, the amplification may be by LAMP , RT-LAMP, NASBA and RPA and EXPAR.

[0099] From another aspect, the present disclosure provides for nucleic acid detection systems. In some embodiments, the systems of the present technology can be used to carry out any of the methods disclosed above to identify one or more target polynucleotide in a sample. The systems of the present technology may comprise any combination of the Cas 12a, the crRNA, PAM duplex, and reporter disclosed above.

[0100] In addition to the components described above, the system may further comprise magnesium salt or a crowding agent as disclosed herein.

[0101] From another aspect, the present technology relates to kits for detecting a target polynucleotide in a sample, wherein the kit comprises a crRNA and a PAM duplex as defined herein. Optionally the kit may further comprise any one or more of the Cas 12a and reporters disclosed herein. The kit may further include instructions for combining the crCRNA and PAM duplex with other elements of the system as described above, as well as with the sample. The instructions may also include instructions relating to the temperature, or duration at which to carry out the methods.EXAMPLES

[0102] The following non-limiting examples are illustrative embodiments and should not be constmed as further limiting the scope of the present invention. These examples will be better understood in combination with the accompanying Figures.Example 1: Materials and MethodsReagents and materials

[0103] All oligonucleotides including the duplex probes used in this work were synthesized by Integrated DNA Technologies (IDT). All modified oligos were purified by HPLC, while unmodified oligos were only subjected to standard desalting. Acidaminococcus sp (AsCasl2a) was purchased from IDT (#10001272). Lachnospiraceae bacterium, LbCasl2a (#M0653T), Casl2 diluent (#B0653A), Casl2 reaction buffer (NEBuffer™ r2.1, #B6002S), and RNase inhibitor (#M0314L) were purchased from New England Biolabs (NEB). Magnesium chloride solution (#7786303) was obtained from Sigma Aldrich. Polyethylene glycol / dimethyl sulfoxide solution 50% (w / v) was purchased from Sigma Aldrich (#P7306). DNase / RNase free deionized water (#10977015) from Thermo Fisher Scientific was used in all experiments. T4 DNA ligase enzyme was procured from Promega (Catalog #: Ml 801).Assembly of nicked DNA activators (Method 1)

[0104] To investigate the effect of nicks on the target strand of dsDNA (target strand: top strand of dsDNA that binds to the gRNA), we assembled three DNA oligonucleotides, designated as NTS, TS Fragment A and TS Fragment B. NTS is the non-target strand while TS is the target strand. DNA oligos were assembled in a IX phosphate-buffered saline solution and heated to 95 °C for 5 min before cooling down to room temperature to facilitate formation of nicked dsDNA. Nicked DNA activators with nicks at 24 different target points were systematically designed by keeping the NTS constant while varying TS Fragments A and B. All sequences are presented in the sequence listing as SEQ ID Nos.: 1 to 50. Additionally, nicks on the NTS were also tested following the same annealing process. Relevant sequences are presented in the sequence listing as SEQ ID NOs.: 51 to 57. We also tested another nick containing dsDNA to establish generality of concept (see the sequence listing, SEQ ID NOs.: 58 to 77).CRISPR-Casl2a assay (Method 2)

[0105] Following the annealing process, CRISPR-Casl2 assay components were prepared in final concentration of IX NEB 2.1 buffer to a final reaction volume of 40 pL, adjusted with nuclease- free water. Final concentrations of assay components were as follows: 50 nM LbCasl2a, 50 nM gRNA, 40 U RNaseinhibitor, 125 nM DNA reporter and 10 nM annealed dsDNA. This mixture was aliquoted in 10 pL volumes into a 96-well plate to enable four technical replicates per condition. Reactions were monitored for 2 hours at 37 °C using the Roche LightCycler 480 II. Fluorescence data at 100 min were normalized and analyzed to assess nicking effects. Sequences of all nucleic acids used are listed in the sequence listing as SEQ ID NOs: 1 to 57. To confirm the generality of our findings, additional experiments employing a different gRNA (gRNA_02, SEQ ID NO: 58) within some regions exhibiting tunable Casl2 activation were conducted using the same protocol.Preparation of nucleic acid targets including RNAs (Method 3)

[0106] All dsDNA were synthesize using gBlock of ssDNA obtained from IDT. T7-containing ssDNA templates for RNA including miRNA and crRNA transcription along with the primers were also purchased from IDT. 40 nM of the T7-containing ssDNA template was amplified using Q5 PCR kit (NEB #M0491) to form the T7-containing dsDNA. RNAs were synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB # E2050) by spiking in the amplified DNA templates into the reaction, and the reaction was performed at 37 °C from 4 to 16 h. The resulting reaction was treated with 4 units of DNase I (NEB #E2050 - M0303AVIAL) for 15 mins at 37 °C before purification with the Monarch RNA Cleanup Kit (NEB #T2040) and quantification with the NanoDrop One spectrophotometer (Thermo Fisher Scientific).Gel electrophoresis (Method 4)

[0107] Cas 12a trans cleavage of <|>X 174 virion DNA in the presence of nicked dsDNA fragments was evaluated by gel electrophoresis. dsDNA fragments with nicks along the target strand, TS_02 -where 02 represent another dsDNA sequence, at positions P8, P12, P13, and P21 were prepared by the annealing process detailed previously. CRISPR-Casl2 assays included 90 nM LbCasl2a, 90 nM gRNA, 20 U RNase inhibitor, 31.25 mM MgC12, <|>X 174 virion DNA (NEB N3023S), and 10 nM of annealed nicked dsDNA all in 20 pL IX NEB r2.1 buffer. The reaction was then incubated for 30 minutes at 37 °C. Reaction mixtures (20 pL) were resolved by 0.8% agarose gels stained with SYBR™ Safe (Thermo Fisher Scientific S33102). Electrophoretic separation was conducted at 120 V for 1 hour and imaging was performed using a Bio-Rad ChemiDoc imaging system. The results of these experiments are presented in FIGs 3B and 3C. All nucleic acid sequences used in these experiments can be found in Table SI, SEQ ID NOs: 1, 2, 17-18, 25-28, and 43-44. Similar experiments were conducted using dsDNA activators with unperturbed target strands (TS_02) and non-target strands nicked at positions S9, Si l and S16 (NTS_02). All sequences for these experiments are listed in the sequence listing as SEQ ID NOs. 1, 51-55.RAPID assay (Method 5)

[0108] ssDNA and RNA Detection: A 50 pL reaction mixture was prepared containing the following components: IX r2.1 NEB buffer, 90 nM LbCasl2a (or AsCasl2a), 50 U RNase inhibitor, 20 nM duplex probe, 90 nM gRNA, 31.25 mM MgC’k 500 nM of a traditional ssDNA reporter (56- FAM / TTATTT / 31ABkFQ; Rl), and 50 nM ssDNA or RNA trigger. From this mixture, 15 pL was aliquoted into a 384-well clear reaction plate (Fisher Scientific 4483285) for technical triplicates. The plate was centrifuged for 2 minutes at 2000 x g and 4 °C using. Following centrifugation, the plate was transferred to a QuantStudio 5 real-time PCR system (ThermoFisher Scientific), where fluorescence was measured over a period of 2 hours at 37 °C.

[0109] dsDNA Detection: A 50 pL reaction mixture was prepared as follows: IX r2.1 NEB buffer, 90 nM LbCas 12a (or AsCas 12a), 50 U RNase inhibitor, 200 nM universal PAM, 90 nM gRNA, 31.25 mM MgC’k 500 nM ssDNA reporter (Rl), 5.81% PEG8000, and 50 nM PAM-free dsDNA trigger. Similar to the ssDNA / RNA detection setup, 15 pL of this reaction mixture was aliquoted into a 384-well clear reaction plate. The remaining steps, including centrifugation and fluorescence measurement, were carried out as described above. All nucleic acid sequences are detailed in in the sequence listing as SEQ ID NOs: 78 to 104.Mismatch detection in ssDNA and miRNA (Method 7)

[0110] All templates containing single or double mismatches in ssDNA were obtained from IDT. A 50 pL reaction mixture was prepared containing the following components: IX r2.1 NEB buffer, 90 nM LbCas 12a, 50 U RNase inhibitor, 20 nM duplex probe, 90 nM gRNA, 31.25 mM MgC’k 500 nM ssDNA reporter (Rl), and 10 nM or 2 pM ssDNA with mismatches or WT. From this mixture, 15 pL was aliquoted into a 384-well clear reaction plate (Fisher Scientific #4483285) for technical triplicates. The plate was centrifuged for 2 minutes at 2000 x g and 4 °C. Following centrifugation, the plate was transferred to a QuantStudio 5 realtime PCR system (ThermoFisher Scientific), where fluorescence was measured over a period of 2 hours at 37 °C. We also challenged RAPID’s selectivity using relatively higher and lower oligonucleotide concentrations and found similar performance for both point mutations and paired two-base mismatches. RNA mismatches were tested the same way, except 5.81% PEG 8000 and alternative reporter were added into the reaction and AsCasl2a was used instead of LbCasl2a. (SEQ ID NOs.: 105 to 153).[oni] Trans cleavage rate calculation: Background subtracted fluorescent kinetic curves were fit to an exponential association model, following the equation y=A*(l-exp(-k*x)). The rate constant k, representing the trans cleavage rate, is plotted for each condition in triplicate.miRNA-21 detection (Method 8)

[0112] Casl2a reactions were assembled by mixing AsCasl2a and gRNA at final concentrations of 90 nM in IX NEB r2.1 buffer supplemented with 31.25 mM MgC12, 5.81% PEG 8000, and lU / pL murine RNase inhibitor (NEB) . The fluorophore-quencher reporter and duplex probe (dsDNA co-activator) were added to the Casl2 reaction at final concentrations of 500 nM and 20 nM, respectively, along with the various concentrations (at 20 pL) of the miRNA target in a 50 pL reaction volume on ice . Reactions were transferred into optical 384-well clear plates (Fisher Scientific #4483285) and incubated at 37 °C for 2 hours while the FAM fluorescent intensity was measured every minute using the QuantStudio 5 RT-PCR System. All oligos are listed in the sequence listing as SEQ ID Nos: 154 to 177. Limit of detection of miRNA-21 calculated from 3o / S, where o is the standard deviation of the background signal and S is the slope of the fluorescence of target concentration line.Lyophilization (Method 9)

[0113] Master mixes for all reaction components including Casl2 proteins, guide RNAs, reporter probes, buffers and salts were prepared as described above, excluding the sample (either miRNA or RT-LAMP amplicons as applicable) and PEG for miRNA reactions. 25 mg / mL each of sucrose and dextran were added to all freeze-dried reactions as a lyoprotectant as described previously. Reactions were flash frozen in liquid nitrogen and freeze-dried overnight in a Labconco FreeZone 6 Liter -84 °C Console Freeze Dryer (catalog #710611200) and vacuum packed with desiccant for storage.RT-LAMP assay (Method 10)

[0114] RT-LAMP reactions using the WarmStart LAMP 2X Master Mix (E1700S, NEB) were assembled in 30 pL containing 0.2 pM for F3 and B3 primers, 1.6 pM for FIP and BIP primers, 0.4 pM for LF and LB primers, and 1.0 pL of template (water, RNA extracted, or in vitro transcribed RNA was used per reaction. These primers targeted the nucleocapsid protein (SEQ ID NOs.: 178 to 195). All reactions were prepared on ice, incubated at 61 °C for 30 minutes, then inactivated at 80 °C for 5 minutes.RT-LAMP / RAPID reaction for SARS-CoV-2 detection (Method 11)

[0115] After the RT-LAMP reaction, 25 pL of the reaction mixture was added to the RAPID reaction mix to achieve a final volume of 50 pL. The final concentrations of the components in the RAPID-LAMP reaction were as follows: IX r2.1 NEB buffer, 90 nM LbCasl2a, 50 U RNase inhibitor, 20 nM PAM duplex 1, 20 nM PAM duplex 2, 90 nM gRNAl, 90 nM gRNA2, 31.25 mM MgCk 500 nM ssDNA reporter , and 25 pL of the RT-LAMP assay, making up the final volume of 50 pL.

[0116] From this mixture, 15 pL was aliquoted into a 384-well clear reaction plate (Fisher Scientific #4483285) in technical triplicates. The plate was centrifuged for 2 minutes at 2000 x g and 4 °C. Following centrifugation, the plate was transferred to a QuantStudio 5 real-time PCR system (ThermoFisher Scientific), where fluorescence was measured over a period of 2 hours at 37 °C.Viral RNA extraction and RT-qPCR for SARS-CoV-2 detection (Method 12)

[0117] Nasopharyngeal swab specimens were tested for positivity and analyzed by RT-qPCR, according to a protocol established by the U.S. Centers for Disease Control and Prevention (CDC) (Ct value < 40 was determined to indicate a positive sample) [3] . Viral RNA was isolated from patient samples using QIAamp Viral RNA Mini Kit (Qiagen, 52906) and RT-qPCR reactions were conducted using the QuantiNova Probe RT-PCR Kit (Qiagen, 208354) according to the manufacturer’s instructions. Briefly, 10 pL reactions were prepared in a 384-well plate format. Primers and probes for SARS-CoV-2 N and RNase P genes are listed in Table 1 below and in the sequence listing as SEQ ID NOs: 196- 201 and were synthesized by IDT. All reactions were performed using the Applied Biosystems QuantStudio 5 Real-Time PCR Systems (Applied Biosystems, USA).

[0118] Table 1: Synthetic RNA controls for molecular reactions. Target synthetic RNA controls were obtained from Twist Bioscience.Patient sample collection and ethical statement (Method 13)

[0119] Nasopharyngeal swabs were obtained from 25 suspected individuals with respiratory disease from the clinical diagnostics lab of Mount Sinai Hospital (MSH), Toronto, Canada. This study was approved by the University of Toronto research ethics board (under human ethics protocol number 39531, which permitted the screening of SARS-CoV-2 vims samples. All experiments were conducted in accordance with relevant regulations and guidelines.Nicked DNA Repair (Method 14)

[0120] DNA fragments corresponding to various nicked positions were prepared by annealing an equimolar mixture of three complementary fragments. The annealing was conducted at 95°C for 4 minutes in an annealing buffer [20 mM Tris-HCl (pH 7.5), 150 mM KC1, 1 mM EDTA, and 50 mM MgC12], followed by a gradient cooling step to 4°C at a rate of 0.1 °C / s. A 50 pL reaction mixture was prepared containing 1 x T4 DNA ligase buffer, 1.5 U of T4 DNA ligase enzyme, 90 nM LbCasl2a, 90 nM Lb gRNA, 50 U RNase inhibitor, DNA reporter , and 50 nM of the annealed DNA fragments. From this mixture, a 15 pL aliquot was dispensed into a clear-bottom black microplate for triplicate fluorescence readings. Trans cleavage activity was monitored at 37°C for 60 minutes using a BioTek plate reader, as previously described. miRNA-DNA Ligation (Method 15)

[0121] For miRNA-DNA ligation, two DNA fragments and the miRNA target were annealed under the same conditions described above for nicked DNA repair. The reaction components and conditions were identical to the nicked DNA repair protocol. For both reactions, the T4 DNA ligase enzyme was excluded in conditions where ligation was not required. This method allowed for precise monitoring of trans cleavage activity under ligation and non-ligation conditions, enabling the evaluation of nick repair and miRNA detection.Example 2: Relationship between single-stranded breaks on CRISPR-Casl2a target sequence and enzymatic trans cleavage activation.

[0122] A systematic investigation of the relationship between single-stranded breaks across the CRISPR-Cas 12a target sequence and enzymatic trans cleavage activation was first conducted, with the findings informing RAPID’s design and ability to overcome canonical Casl2a PAM and protospacer structural requirements. Traditionally, Casl2a introduces a staggered dsDNA break on the target nucleic acid strand when it is activated (FIG. 1A).

[0123] In the present experiments, the dynamic interactions between CRISPR-Cas 12a and single-stranded breaks within the PAM and protospacer was studied by introducing single-stranded breaks, to the target strand of dsDNA (Fig. IB, pink triangle). In the system illustrated in FIG. IB, the sequence recognized by Casl2a contains 24 possible nick sites, starting from site between the first two PAM bases, denoted as Pl, to the gRNA terminus adjacent nick, P24 (Methods 1&2). Our study revealed distinct patterns in Casl2a activation, influenced by the positioning of the single stranded break (Fig . 1 C &D) . Certain target sequencediscontinuities elicited full activation of the Cas 12a protein, others achieved partial activation (semi-muted), and some were incapable of triggering the Cas 12a nuclease effector (muted).

[0124] Subsequently, six experimental configurations, designated as Types I through VI, were engineered each designed to interrogate specific aspects of Cas 12a’s interaction with structurally diverse ssDNA and dsDNA substrates (FIG. 2A). The Type I configuration consists of dsDNA, wherein the non-target strand is unperturbed, and the target strand is incrementally extended from Pl to P24 to fulfill pairing interactions between the target strand and gRNA. The objective was to ascertain the minimal target strand length necessary to trigger Cas 12a activation, revealing that activation commenced at P18 on the target strand for Type I (FIG. 2B). Conversely, in Type II, the target strand was elongated from the distal end of the PAM region (P24) towards the PAM site (Pl), with activation detected when the target strand is extended to P6 or further. Types III and IV mimic the configurations of Types I and II, respectively, but with the non-target strand omitted to assess the influence of ssDNA on Cas 12a activation. Our findings showed activation when the target strand is extended beyond P21 for Type III and when the target strand is extended from P24 to P7 or further for Type IV. Types V and VI systems display nicked ssDNA and dsDNA configurations, respectively. Type VI, is comprised of a dsDNA target, while Type V is comprised solely of a ssDNA target strand, both featuring single nucleotide nicks across positions Pl to P24 on the target sequence. In Type V, Cas 12a activation was either completely or partially impaired across several segments, notably from P7 to P17 and P19 to P20, when compared to Type VI. This pattern of deactivation, which was found to correspond to regions that interact with the endonuclease domain of Cas 12a demonstrate that the presence of nicks may perturb protein-DNA interaction dynamics. The activation patterns across these configurations are reported in the heatmap of FIG. 2B. All the nucleic acid sequences are presented as SEQ ID NOs: 1 to 50.

[0125] Expanding our investigation, we picked specific locations of Type VI and compared the trans cleavage fluorescence and agarose gel results (FIG. 3 A, Method 3). Similar nicks were introduced to the non-target strand of the dsDNA as illustrated in FIG. 3D, (SEQ ID NOs.: 52 to 57). Interestingly, these nicks did not influence activation of the Cas 12a protein FIG. 3C and 3D, underscoring the importance of the target strand nicks in modulating Cas 12a activation. Specifically, full activation of the enzyme was observed at positions Pl through P8, PIO, and from P15 through P18, as well as at positions P21 to P24 (FIGs. 2B, and 3D). Interestingly, positions P9, Pl 1, P13, P19, and P20 only exhibited partial activation, whereas nicking at positions P12 and P14 completely disabled Casl2a trans cleavage activity (FIGs. 2B, and 3D). This phenomenon highlights the potential for the modulation of Cas 12a activation by the strategic introduction of single-stranded breaks.

[0126] To validate the generality of our findings, we expanded our study to include a unique spacer sequence as depicted in SEQ ID. NO.: 58 to 77, focusing on positions known to exhibit significant activation variability (P5, P8, P9, PIO, Pl 1, P16, P20, and P22) between the various configurations. The results are shown inFIG. 3E, and the DNA sequences are presented as SEQ ID. NOs: 58 to 77. The outcomes were consistent with our initial observations across all six configurations (Types I to VI), supporting the potential universality of our findings related to Casl2a activation.

[0127] To further understand the mechanism of deactivated Casl2a upon nick introduction, the local protein environments at the nicking positions from the published protein crystal structures of Cpfl in complex with gRNA and dsDNA was approximated. It was hypothesized that nicks at certain positions may alter protein- DNA interactions, thereby affecting the enzymatic activation of Casl2a. Table 2 summarizes the nick positions rendering substantially diminished activity of Cas 12a, along with their interacting amino acids (within 3.5 angstroms). Notably, the segments from P9 to P14 in this work, which exhibited muted Casl2 activation, interact with the endonuclease domain (RuvC and BH), which is critical for both cis and trans cleavage. P19 and P20, exhibiting semi-muted activation upon nick introduction, primarily interact with amino acids in the REC2 domain, which is involved in gRNA binding to the protein. This suggests that modifications at these sites could potentially influence the gRNA-protein binding efficiency.

[0128] These observations provide insight into the complex mechanisms regulating Casl2a activation and offer a novel perspective on the strategic manipulation of CRISPR systems at the molecular level.

[0129] Table 2: Structure of AsCasl2 in complex with gRNA and target DNA. PDB ID: 5B43Example 3: Development of the PAM duplex

[0130] We next leveraged our findings from the six configurations to develop the PAM-free CRISPR-Casl2a ssDNA / dsDNA / RNA RAPID detection platform (Method 4-5).

[0131] We assessed Cas 12a activation across the 24 nicked positions (Pl to P24) for configurations Types I through VI to identify an optimal nicking point for sensor development (FIG. 2B). The Type VI system with a nicking at P8 emerged as a promising candidate, as nicking at this point produced complete activation of the Type VI system and negligible activation of Types I to V. This unique configuration allows for the development of an "AND logic" sensing mechanism within the RAPID system, which requires complete system assembly for Cas 12a activation. Consequently, the design of the RAPID sensors was focused on the Type VI configuration, referred to as RAPID Type VI.

[0132] As shown in FIG. 4, the RAPID Type VI system is highly programmable, as enzyme activation can be triggered by target ssDNA, RNA, and dsDNA. CRISPR gRNAs were engineered for sequence-specific binding of these diverse nucleic acid types, resulting in sequence responsive trans cleavage of a fluorescence reporter. Another critical component of RAPID is the PAM duplex (FIG. 4A), a dsDNA sequence consisting of a PAM and neighbouring ssDNA toehold domain (also referred to as a PAM-duplex with sticky ends). The PAM sequences are introduced in trans configuration, meaning PAM sites are separate from the target nucleic acid. Short ssDNA or miRNA targets can bind to the toehold domain, creating a pseudo-nick corresponding to position P8 in the Type VI system, thus enabling Casl2a activation (FIG. 4A - I). Similarly, longer ssDNA or RNA sequences can also trigger system activation (FIG. 4A - II). Moreover, we have designed a universal PAM duplex that lacks a toehold domain (also referred to as a blunt PAM duplex), allowing for the detection of PAM-free dsDNA (as depicted in FIG. 4A - III and IV). In this configuration, the PAM sequence is again introduced in trans and is structurally distinct from the dsDNA trigger. Herein, we present two scenarios for dsDNA detection: I) The gRNA-Casl2a complex binds to both the universal PAM duplex and the blunt-ended PAM-free dsDNA, leading to activation (FIG. 4A - III), and II) A segment of the gRNA binds to the universal PAM while another segment binds to an extended PAM-free dsDNA target, thereby activating Casl2a (FIG. 4A - IV). As such, RAPID can directly detect PAM- free dsDNA in multiple configurations.Example 4: Nucleic acid detection with RAPID

[0133] RAPID was next tested using two commercially available Cas 12a orthologs, specifically Acidaminococcus sp. (AsCasl2a) and Lachnospiraceae bacterium (LbCasl2a). The initial experiments focused on enhancing detection sensitivity of RAPID for ssDNA by pairing ssDNA with sticky end dsDNA containing the PAM duplex, as depicted in FIG.4A-I, Method 5. Trans cleavage signal enhancement of CRISPR-Cas 12 is based on the premise that dsDNA significantly amplifies the trans cleavage signal compared to ssDNA. Therefore, we experimented with short ssDNA target strands (light blue) of 28 nucleotides (nt), corresponding to FIG. 4A-I, and longer strands of 89 and 139 nt, corresponding to FIG. 4A-II. Utilizing PAM-containing dsDNA as a benchmark, we observed that the trans cleavage signals from all ssDNA triggers were comparable to those from PAM-containing dsDNA for LbCasl2a and AsCasl2a (FIGB). Moreover, it was observed that AsCasl2a exhibited a higher no template control (NTC) background signal than that of LbCasl2a.

[0134] Extending the analysis to RNA triggers, differential trans cleavage activation thresholds were found between the two Cas 12a variants. RNA target sequences activated trans cleavage remarkably well when complexed with AsCasl2a, while trans cleavage was significantly muted with LbCasl2a (FIG. 4C). The target RNA lengths tested included a 28-nt sequence, akin to the setup in FIG. 4A-I, and longer sequences of 65 and 116-nt, paralleling the setup in Fig. 4A-II. Notably, the shorter target RNA (28-nt miRNA) was more effective at triggering trans cleavage than longer RNA counterparts.

[0135] Further exploration of the RAPID system revealed efficient detection of PAM-free dsDNA. By designing a blunt dsDNA alongside a universal PAM duplex (FIG. 4A-III), we achieved system activation through the partial binding of the blunt target dsDNA and the universal PAM duplex to the gRNA (FIG. 4D). The effect of the universal PAM duplex concentration was experimentally studied for both blunt-ended and longer dsDNA (FIGs. 4D and 4E), and the optimal concentration was determined to be 200 nM. Leveraging this optimized universal PAM concentration, we assessed detection of PAM-free dsDNA at an arbitrary location, mirroring the configuration in FIG. 4A- IV for a longer target sequence. RAPID demonstrated effective detection of PAM-free dsDNA, as presented in FIG. 4F, with the performance of PAM-free dsDNA detection on par with that of PAM-containing dsDNA (FIGs. 4G and 4H). This establishes RAPID as a versatile and universal platform for PAM-free detection of nucleic acids.Example 5: Detection of single and two-point mutations with RAPID

[0136] The capacity to identify point mutations in target ssDNA and RNA sequences can provide important advantages to diagnostics efforts, including enabling monitoring of viral variants, the presence of drug resistance, or the detection of oncogenic mutations, among many other applications. Detecting subtle differences within nucleic acid sequences is currently a challenge for nucleic acid diagnostics. To investigate the potential of the RAPID platform to provide single nucleotide polymorphism (SNP) detection, we screened ssDNA sequences containing point mutations and paired two-base mismatches across the target binding regions (1-16, FIGs 5 A -5 C) . The sequences, including the target regions (bold) and the mismatches (underlined), are detailed in the sequence listing as SEQ ID. NOs.: 105 to 153, facilitating a direct comparison between the mutated sequences and their wildtype (WT) counterparts. RAPID demonstrated a marked ability to distinguish point mutations located proximal to the trans PAM region for ssDNA (positions #1 to 10) termed "RAPID seed region" (FIG. 5 A, Method 7). However, the detection efficiency waned for mutations situated more distal from the trans PAM (positions #11 to 16). Next, we introduced paired two-base DNA mismatches within the target sequences (FIG. 5B). As above, we saw strong selectivity in the RAPID seed region. With the capacity to detect sequences in a PAM-free manner, we can rationally design detection schemes to place target mutations within the region of high selectivity for both SNP and paired mismatches. Importantly, these extended regions of specificity for SNPs and two-paired mismatches are not possible using the conventional CRISPR-Casl2a system containing ssDNA. Using RAPID, the duplex probe enables the formation of dsDNA to enhance discrimination towards point and paired two base mismatches.

[0137] We performed further tests to assess RAPID’s sensitivity to point mutations using synthetic miRNA-21 as a model target (FIG. 5C) combined with a unique reporter developed to enhance the performance of the RAPID system. Contrary to the trend observed for RAPID Casl2a / ssDNA systems, RAPID’s selectivity toward point mutations in miRNA found that certain locations within the distal region of the miRNA targetalso exhibited a relatively high degree of selectivity for point mutations similar to some positions closer to the trans PAM region. This distinction reveals important insights regarding RAPID’s unique interactions with miRNA and ssDNA.

[0138] From the mutational datasets, we can conclude that Casl2a activation requirements are altered when accommodating non-canonical RNA substrates. Overall, RAPID succeeded at identifying point mutations in ssDNA and miRNA and selectivity thresholds were appropriate for diagnostic applications. RAPID’s sensitivity to nucleotide alterations (SNPs) parallels findings from recent studies employing Casl2a systems for RNA detection [2] . Additionally, RAPID covers 16-nt of the target sequence and is specific over a longer region compared to the recent studies that could only detect 12-nt of the target sequence with limited specificity [2] . All data was collected as a time series. The trans cleavage rate data was analysed within 2 h (Method 7), Similar results were observed at a 60-minute end point. The data is presented in FIGs. 6A-6C.Example 6: miRNA detection with RAPID

[0139] miRNAs are a category of small, single-stranded, non-protein-coding RNAs with a length of approximately 19-23 nucleotides, and have been widely reported as biomarkers of several cancers, diabetes, immune dysregulation, and other disease states. The direct detection of these biomarkers using conventional Casl2a systems is challenging due to incompatible reaction buffers, the requirement for a PAM, and high background. Leveraging the new characterized chimeric reporter, we optimized the RAPID system to directly target miRNAs by identifying the optimal enzymatic concentrations and buffer conditions. Based on our earlier results showing AsCasl2a has a higher performance for RNA targeting (FIG. 4C), we chose to test AsCasl2a in these experiments .

[0140] We began with an evaluation of MgC12 concentration, which we found was optimal at 31 mM. (FIG. 7A). Next, a titration of duplex DNA, AsCasl2a enzyme, and gRNA concentrations were screened (FIG. 7B), finding optimal performance at approximately 20 nM, 90 nM, and 90 nM, respectively (heatmap - FIG. 7C). Finally, we found that the addition of ~6% PEG-8000 as a crowding agent enhanced the trans cleavage signal (FIG. 7E).

[0141] With the optimized conditions in place, we proceeded with the development of a RAPID assay for miRNA- 21 (FIG. 7E), chosen for its significance in breast cancer prognosis (Method 8). Beginning with the use of RAPID alone, we found a detection limit of 97 pM (Method 8, FIGs. 7F and 7G), which provides comparable performance to Casl3 RNA-based miRNA detection. This enhancement is particularly notable given that RAPID covers a 16 nt miRNA sequence, compared to the <12 nt coverage in earlier Casl2 systems. Additionally, we tested RAPID’s specificity for miRNA-21 against miRNA-210 and miRNA-320, which are biomarkers for other conditions, including cancer prognosis — and confirmed orthogonality in detection (FIGs. 7H and 71).

[0142] The isothermal nature of the RAPID platform positions it well as a tool for decentralized diagnostics, so we evaluated the performance of freeze-dried reagents for the potential distribution of test kits without a cold chain (Method 9). As can be seen from endpoint reads (2 h), we found that the system demonstrated robust stability for up to one week from the time of freeze-drying (FIG. 7J and 7K).Example 7: RAPID coupled with RT-LAMP for SARS-CoV-2 RNA detection in clinical samples

[0143] With the goal of demonstrating the capacity for RAPID to serve as a diagnostic platform for infectious disease, we developed a RAPID assay for the RNA genome of SARS-CoV-2 with the aim of providing comparable sensitivity to the gold standard reverse-transcription quantitative PCR (RT-qPCR). To achieve this level of sensitivity, we integrated the RAPID platform with the isothermal method RT-LAMP; (FIG. 8A, Method 10). Priorto coupling RAPID with RT-LAMP, we first tested RAPID separately with synthetic DNA strands that mimic the two loops of the anticipated LAMP dumbbell amplicon structure (FIG. 8B and 8C)). The results revealed that designing the system to target both ssDNA LAMP dumbbells could significantly enhance detection. We moved forward with an integrated RT-LAMP / RAPID configuration, where we strategically targeted the loop region of the LAMP-generated dumbbell structure as the CRISPR gRNA target regions (FIG. 8B) (Method 11). Furthermore, the use of the trans-acting PAM duplex sequence as a feature of the RAPID system was used to boost the CRISPR / Cas 12a cleavage of the reporter probe by transitioning the detection ssDNA region to dsDNA, as shown by the CRISPR / Cas 12a plus gRNA complex.

[0144] We validated the novel combined RT-LAMP and RAPID assay design using SARS-CoV-2 RNA (nucleocapsid gene) and found that detection of SARS-CoV-2 positive patient samples was achieved within 5 minutes following a 30 min RT-LAMP pre-amplification reaction (FIG. 8D, SEQ ID NOs: 178 to 195). We further determined that the addition 25 pL of the RT-LAMP reaction volume did not inhibit the RAPID reaction (FIG. 8E), and so this volume was chosen for further experiments.

[0145] Having established RAPID for viral RNA detection, we next evaluated the analytical sensitivity of RT- LAMP / RAPID. In sensitivity assessments, the coupled RT-LAMP / RAPID system detected viral concentrations as low as 1 copy / pL (FIG. 8F and 8G), comparable to standard RT-qPCR methods. Further testing confirmed RAPID's ability to detect synthetic RNA corresponding to SARS-CoV-2 variants of concern (VOCs), including alpha, beta, gamma, delta, and omicron, as well as the original Wuhan strain (FIG. 8G, and 8H, Table 1).

[0146] A selectivity study reinforced these findings, with RAPID achieving high specificity for detecting only SARS-CoV -2 when tested using synthetic RNAs from other viral respiratory pathogens, including influenza virus (H1N1 and H7N9) and other coronaviruses (229E, NL63, OC43, MERS-CoV, and SARS-CoV-1) (FIG. 81 and 8J). Briefly, each synthetic RNA consists of non-overlapping fragments generated from DNA fragments and then transcribed into ssRNA, providing coverage of greater than 99.9% of the bases for each respiratory pathogen.

[0147] We next set out to evaluate the diagnostic performance of RT-LAMP / RAPID using clinical samples from suspected cases of SARS-CoV-2 infection collected during the pandemic in Toronto, Canada. (FIG. 8A, Methods 11 - 12) . A total of 25 nasopharyngeal samples were analysed using the RAPID diagnostic platform in parallel with RT-qPCR (U.S. CDC protocol), with RAPID, in general, providing comparable accuracy to the gold standard method (FIG. 9A, SEQ ID NOs: 196 to 201).

[0148] Table 3: Comparison of SARS-Cov2 patient sample data with RAPID and RT-qPCR.

[0149] Of the 25 patient samples tested for SARS-CoV-2, four samples with Ct values >33 demonstrated false negative, leveling up accuracy at 84.00% (95% confidence interval [CI], 63.92% to 95.46%) (Table 4). However, for samples with Ct values <33, the RT-LAMP / RAPID was 100% (95% CI, 83.89% to 100.00%) accurate compared to RT-qPCR (Table 5), supporting a role for RAPID in providing isothermal and fastscreening of viral RNA targets. To ensure the RNA integrity of the patient samples and as a criterion for sample inclusion in this phase of the project, patient RNaseP measurement was performed using RT-qPCR (FIG. 9B), with Ct values for RNaseP ranging from 28.8 to 37.2 (Table 3).

[0150] Table 4: Patient samples (Ct value <40).* To check the calculations, visit: https: / / www.medcalc.org / calc / diagnostic test.php

[0151] Table 5 : Table of statistical analyses at a 95% confidence interval for clinical sensitivity, specificity, and disease prevalence, highlighting that RAPID achieved 100% accuracy in samples with a cycle threshold (Ct) value <33.

[0152] Freeze-dried RAPID was also performed for the individual Casl2a and RT-LAMP reaction components stored at room temperature (FIGs. 9C-9E). We tested the system stability up to one week, with the 2-hour endpoint reported (37 °C). The results were comparable to previously reported CRISPR-based freeze- drying. It is believed that by using commercial lyophilization processes to produce freeze-dried pellets and provide more effective packaging methods, the combined system has the potential for deployment as a decentralized medical diagnostic.Example 8: Ligation-Induced Trans Cleavage

[0153] As discussed above our results demonstrated that nicked DNA exhibits varying activation patterns depending on the position of the nick. In the following experiments, we demonstrated that in situ ligation of the nicked DNA can fully restore trans cleavage activity. As shown in FIG. 10A, the T4 DNA ligase was used to ligate the nick in the system shown at various positions. In these experiments, the 5' end of the nicked DNA was phosphorylated, to enable repair in the presence of ligase. As best seen in the bar graph,the presence of the ligase restores the trans cleavage signal at position Pl 2. Without the ligase enzyme, no trans cleavage is observed. The heat map provided in FIG. 10B further illustrates that positions P5 and P8 have complete activation regardless of ligase presence, corresponding to the data shown in FIG. 2B. As previously shown, position P9 exhibits partial activation without ligase, which is now shown to be fully restored upon ligase addition. At position P12, no activation occurs without ligase; however, the signal is now shown to be recovered with ligase. In these experiments, dsDNA served as a control template without a nick, and NTC represents the no-target control.

[0154] Similar experiments were conducted with miRNA targets. As seen in FIG. 10C, in the presence of the miRNA target, a signal was generated, whereas no signal was detected in the negative control lacking the miRNA target. The ability of ligation to restore trans activity of Casl2a in the detection of miRNA with the “nick” created at various positions (P5, P8, P9, P12, and NTC) was then tested. As seen in the FIG. 10D &10E the trans activity was recovered at position P8. This approach is particularly advantageous for miRNA detection, highlighting the potential of ligation-based activation in diagnostic applications.REFERENCESThe following documents are incorporated herein by reference in their entirety.1. Chen, J. S.; Ma, E.; Harrington, L. B.; Da Costa, M.; Tian, X.; Palefsky, J. M.; Doudna, J. A. Science. 2018, 360, 436-439.2. Santosh R. Rananaware, Emma K. Vesco, Grace M. Shoemaker, Swapnil S. Anekar, Luke Samuel W. Sandoval, Katelyn S. Meister, Nicolas C. Macaluso, Long T. Nguyen & Piyush K. Jain. Nature Communications. 2023, 14, 5409.3. Centers For Disease Control And Prevention. Centers For Disease Control and Prevention. 2020

Claims

CLAIMS1. A method for detecting a target polynucleotide in a sample comprising: a) contacting the sample with: i) a Casl2a CRISPR-associated (Cas) enzyme; ii) a CRISPR RNA (crRNA) comprising a guide sequence, the guide sequence having a protospacer adjacent motif (PAM)-proximal portion and a PAM -distal portion, the PAM- distal portion being configured to hybridize with the target polynucleotide; iii) a PAM duplex having a PAM sequence and a target strand configured to bind to the PAM-proximal portion of the guide sequence, such that upon binding to the guide sequence, a distal end of the target strand of the PAM duplex and the target polynucleotide are spaced apart from one another by at least one nucleotide, and a trans nuclease activity of Cas 12a is activated; and iv) a reporter capable of being activated by the trans nuclease activity of Cas 12a; and b) measuring a detectable signal produced by the activated reporter, wherein said measuring provides for the detection of the target polynucleotide in the sample.

2. The method of claim 1, wherein the at least one nucleotide is one nucleotide.

3. The method of claim 1, wherein the at least one nucleotide is two nucleotides.

4. The method of any one of claims 1 to 3, wherein the at least one nucleotide, is an at least one mismatched nucleotide between the PAM-distal portion and the target polynucleotide.

5. The method of any one of claims 1 to 4, wherein the target strand of the PAM duplex binds to the the first four nucleotides of the PAM-proximal portion of the guide sequence.

6. The method of claim 5, wherein the target strand of the PAM duplex binds to the first four nucleotides of the PAM-proximal portion of the guide sequence.

7. The method of any one of claims 1 to 6, wherein the target nucleotide sequence is a PAM-free single stranded DNA, a PAM-free double-stranded DNA, or an RNA.

8. The method of claim 7, wherein the RNA is a miRNA.

9. The method of any one of claims 1 to 7, wherein the target nucleotide sequence is a virus-associated polynucleotide.

10. The method of claim 9, wherein the virus-associated polynucleotide is SARS-CoV-2 polynucleotide, a hepatitis C virus (HCV) polynucleotide, a human immunodeficiency virus (HIV) polynucleotide, a malaria polynucleotide, or a bluetongue virus polynucleotide.11 . The method of any one of claims 1 to 10, wherein the PAM duplex is blunt ended.

12. The method of any one of claims 1 to 10, wherein a non-target strand of the PAM duplex is longer than the target strand.

13. The method of claim 12, wherein the target polynucleotide is a ssDNA or RNA, and the non-target strand of the PAM duplex is configured to bind to a portion of the target polynucleotide which is not bound to the PAM-distal portion of the guide sequence.

14. The method of any one of claims 1 to 13, further comprising amplifying the target polynucleotide in the sample.

15. The method of claim 14, wherein the amplification is by LAMP, RT-LAMP, NASBA, RPA, and EXPAR.

16. The method of any one claims 1 to 15, further comprising contacting the sample with a DNA ligase (e.g. T4 DNA ligase) in step a).

17. A nucleic acid detection system for detecting a target polynucleotide in a sample comprising: i) a Casl2a CRISPR-associated (Cas) enzyme; ii) a CRISPR RNA (crRNA) comprising a guide sequence, the guide sequence having a protospacer adjacent motif (PAM)-proximal portion and a PAM-distal portion, the PAM-distal portion being configured to hybridize with the target polynucleotide; iii) a PAM duplex having a PAM sequence and a target strand configured to bind to the PAM-proximal portion of the guide sequence, such that upon binding to the guide sequence, a distal end of the target strand of the PAM duplex and the target polynucleotide are spaced apart from one another by at least one nucleotide, and a trans nuclease activity of Cas 12a is activated; and, iv) a reporter capable of being activated by the trans nuclease activity of Cas 12a.

18. The system of claim 17 wherein: a) the at least one nucleotide is as defined in any one of claims 2 to 4; b) the target strand of the PAM duplex binds to the guide sequence as defined in claim 5 or 6; c) the target polynucleotide is as defined in any one of claims 7 to 10; d) the PAM duplex is as defined in any one of claims 11 to 13; or e) any combination of a) to d).

19. The system of claim 17 or 18, further comprising a DNA ligase (e.g. a T4 DNA ligase).

20. A kit for detecting a target polynucleotide in a sample, the kit comprising the crRNA as defined in claim 1 and a PAM duplex as defined in in any one of claims 1, and 12 to 14.21 . The system of claim 17 to 19, or the kit of claim 20 for use in detecting : a) a single or a two-point mutation in a target polynucleotide; b) a miRNA; or c) a virus-associated polynucleotide.

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