Multi-omics assay and uses thereof

Abstract

Provided herein are methods and materials using multi-omics assay to detect DNA, RNA, and / or polypeptides. In some cases, the methods and materials provided herein can be used for simultaneous detection of one or more target analytes (e.g., a target gene of interest) in a sample. In some cases, the outputs generated are standardized into a common data format, thereby allowing integrated processing, interpretation, and comparison across multiple different types of analytes (e.g., biomolecular classes). For example, the method includes simultaneous detection of DNA, RNA, and / or polypeptides in a single sample.

Description

[0001] Attorney Docket No. 15670-0433WO1 (SD2025-050-1)

[0002] MULTI-OMICS ASSAY AND USES THEREOF

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 692,387, filed on September 9, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing that has been submitted electronically as an XML file named “15670-0433W01_SL_ST26.XML.” The XML file, created on September 8, 2025, is 6,312 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0007] TECHNICAL FIELD

[0008] The present disclosure relates to methods and materials of using multi-omics assay to detect DNA, RNA, and / or polypeptides.

[0009] BACKGROUND

[0010] Traditional methods for profiling DNA, RNA, and proteins, such as sequencing, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and chromatography, each require a unique set of components and reagents. For example, DNA and RNA profiling needs primers, polymerases, and various devices for experimentation and signal readout. Similarly, protein assays require antibodies, reagents, and optical instruments. These methods are often timeconsuming and resource-intensive. Therefore, novel detection methods are needed that reduce consumable and high instrument costs without compromising sensitivity and specificity.

[0011] SUMMARY

[0012] The present disclosure provides methods and materials for simultaneous detection of one or more target analytes in a sample. For example, this document provides methods and materials that relate to a multi-omics assay that can simultaneously detect DNA, RNA, and / or polypeptides in a sample. As demonstrated herein the methods and materials provided herein require minimal changes in Attorney Docket No. 15670-0433WO1 (SD2025-050-1) components to simultaneously profile nucleic acid molecules and polypeptides with high sensitivity and specificity, without the need for amplification.

[0013] Provided herein are multi-omics methods for simultaneously determining the presence of one or more target analytes in a sample, the method including (a) isolating one or more analytes from the sample and converting the one or more analytes into one or more target molecules, (b) contacting the one or more target molecules with an activatable Cas construct and a cleavable probe, (c) measuring a signal readout from the Cas-mediated collateral cleavage of the cleavable probe, and (d) determining the presence of the one or more target analytes in the sample.

[0014] In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the biological sample includes a tissue, a cell, a body fluid, or a biopsy sample. In some embodiments, the environmental sample includes soil, water, sediments, ice, biofilms, waste materials, or rocks. In some embodiments, the biological sample is blood.

[0015] In some embodiments, the method provided herein further includes processing the sample. In some embodiments, processing the sample includes separating plasma from blood. In some embodiments, the one or more analytes include a DNA molecule, an RNA molecule, and / or a polypeptide. In some embodiments, converting one or more analytes includes reverse transcribing an RNA molecule into a cDNA molecule. In some embodiments, converting one or more analytes includes contacting the polypeptide with pair of DNA-conjugated antibodies forming a DNA-tagged antibody-polypeptide complex. In some embodiments, the pair of DNA-conjugated antibodies includes a primary antibody with a DNA tag and a secondary antibody with a DNA tag forming the DNA-tagged antibody-polypeptide complex.

[0016] In some embodiments, the antibody includes a monoclonal antibody, a polyclonal antibody, or a nanobody. In some embodiments, the antibody includes a fully split antibody, a partially split antibody, or a no-split antibody. In some embodiments, the fully split antibody includes a Fab fragment, an F(ab’)2 fragments, or a single-chain variable fragment (scFv). In some embodiments, the partially split antibody includes a half-antibody, a heavy chain-only antibody, or an Fc fragment. In some embodiments, the DNA tag on the primary antibody bound to the DNA-tagged antibody-polypeptide complex is captured on a DNA-fimctionalized surface. In some Attorney Docket No. 15670-0433WO1 (SD2025-050-1) embodiments, the one or more target molecules include a cDNA molecule, a DNA molecule, or the DNA-tagged antibody-polypeptide complex.

[0017] In some embodiments, the cleavable probe is a nucleic acid hybrid probe. In some embodiments, the nucleic acid hybrid probe includes a chimera probe, a fragment of a single-stranded DNA or RNA, a double-stranded DNA or RNA, or a partially double-stranded DNA or RNA. In some embodiments, the cleavable probe includes an enzyme-cleavable probe, a photolabile-cleavable probe, or a pH-cleavable probe. In some embodiments, the chimera probe includes both DNA and RNA bases within a single strand. In some embodiments, the nucleic acid hybrid probe includes a donor fluorophore and an acceptor fluorophore. In some embodiments, the Cas construct includes a guide RNA and Cas 12 and / or Cas 13. In some embodiments, contacting includes binding of the Cas construct to the one or more target molecule.

[0018] In some embodiments, contacting includes binding of the Cas construct to the DNA tag on the secondary antibody bound to the DNA-tagged antibody-polypeptide complex. In some embodiments, the signal includes an optical signal, an electrical signal, an electrochemical signal. In some embodiments, the signal readout includes fluorescence, luminescence, absorption spectroscopy, electrochemical redox reaction, graphene field-effect transistor (gFET) sensor output, capacitance, resistance, or conductance. In some embodiments, the methods further includes amplifying the signal by polymerase chain reaction (PCR), increasing time or temperature, adding an additional Cas construct, adding an additional cleavable chimera probe, adding an additional dsDNA, ssDNA, RNA, cDNA, and / or ssDNA tagged antibody. In some embodiments, the signal readout includes data refinement through noise filtration module, pre-configured analytical algorithm, feature-extraction algorithm, classification of positive versus negative signal, or decision threshold module. In some embodiments, the presence of one or more target analytes in a sample indicates a cancer or an infectious disease.

[0019] In some embodiments, contacting the one or more target molecules with the activatable Cas construct and the cleavable probe is performed in separate vessels for each target molecule. In some embodiments, contacting the one or more target molecules with the activatable Cas construct and the cleavable probe is performed in the same vessel for the one or more target molecules. In some embodiments, Attorney Docket No. 15670-0433WO1 (SD2025-050-1) contacting the one or more target molecules with the activatable Cas construct and the cleavable probe is performed in the same vessel for at least two target molecules.

[0020] In some embodiments, measuring the signal readout from the Cas-mediated collateral cleavage of the probe is performed in separate vessels for each target molecule. In some embodiments, measuring the signal readout from the Cas-mediated collateral cleavage of the probe is performed in the same vessel for the one or more target molecules. In some embodiments, measuring the signal readout from the Cas- mediated collateral cleavage of the probe is performed in the same vessel for at least two target molecules.

[0021] In some embodiments, the signal readout is measured using the same signal for the one or more target molecules. In some embodiments, the signal readout is measured using a distinct signal for each target molecule. In some embodiments, the signal readout is measured using the same signal for at least two target molecules.

[0022] In some embodiments, the presence of the one or more target analytes in the sample is determined in the same vessel. In some embodiments, the presence of the one or more target analytes in the sample is determined in separate vessels for each target analyte. In some embodiments, the presence of the one or more target analytes in the sample is determined in the same vessel for at least two target analytes.

[0023] In some embodiments, the cancer includes a solid tumor or a blood cancer. In some embodiments, the solid tumor includes head and neck squamous cell carcinoma, pancreatic cancer, renal carcinoma, breast cancer, lung cancer, prostate cancer, glioma, melanoma, keratinocyte cancer, ovarian cancer, liver cancer, kidney cancer, bladder cancer, thyroid cancer, sarcoma, stomach cancer, cervical cancer, endometrial cancer, esophageal cancer, thymoma, soft tissue sarcoma, bone cancer, testicular cancer, penile cancer, gallbladder cancer, uterine sarcoma, adrenal gland cancer, ampullary cancer, hepatic angiosarcoma, nasal, or paranasal sinus cancer. In some embodiments, the blood cancer is leukemia, lymphoma, myeloma, myelodysplastic syndrome, or myeloproliferative neoplasm. In some embodiments, the infectious disease includes a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a prion infection.

[0024] In some embodiments, the biological sample is obtained from a subject, where the subject is a mammal. Attorney Docket No. 15670-0433WO1 (SD2025-050-1)

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0026] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0027] DESCRIPTION OF DRAWINGS

[0028] FIGS. 1A-1C. Fluorescence-based detection using Casl2. FIG. 1A: Schematic representation of the fluorescence-based spectroscopic analysis using single-stranded DNA / RNA targets. FIG. IB: Schematic illustration of a 6-FAM-labeled chimera probe, containing both RNA and DNA bases, immobilized on a surface of the sensing element (e.g., a graphene field-effect transistor (gFET) sensor). The system enables detection of target DNA or RNA sequences via Casl2 and / or Casl3 complexes guided by a target-specific crRNA. FIG. 1C: Schematic illustration of the Casl2- based antigen / protein detection strategy. A target-specific antibody is conjugated with a single-stranded DNA (ssDNA) containing multiple Casl2 recognition sites. Upon antigen binding, a secondary antibody bearing the ssDNA probe enables Casl2 activation and collateral cleavage of the chimera probe, resulting in a measurable fluorescence signal.

[0029] FIGS. 2A-2E. Detection of double-stranded DNA (dsDNA) and singlestranded DNA (ssDNA) targets. FIG. 2A: Fluorescence measurements demonstrated a concentration-dependent increase in signal upon detection of BRAF-specific dsDNA. FIG. 2B: ssDNA was tested at varying concentrations. The results showed a clear concentration-dependent fluorescence response compared to the non-target ssDNA control. FIG. 2C: Detection of five different microRNAs was achieved using Casl3a complexed with specific guide RNAs (crRNAs), each showing distinct and targetspecific fluorescence signals. FIG. 2D: The assay demonstrated Casl2a-mediated signal enhancement upon PD-L1 antigen binding. This response was observed only in Attorney Docket No. 15670-0433WO1 (SD2025-050-1) the presence of the specific antibody and conjugated ssDNA on the secondary antibody, whereas the control samples with non-target antibodies showed negligible signal. FIG. 2E: Target miRNA23 was successfully detected compared to the nontarget control (miRNA 159). (No_Abl_Ab2-TDNA: No primary antibody for PD-L1 (CD274)_Secondary antibody specific for PD-L1 (CD274) conjugated with Target ssDNA; Abl_Control@Ab2-TDNA: Primary antibody specific for PD-L1 (CD274)_ Control secondary antibody (not binding to PD-L1) conjugated with Target ssDNA; Abl_Control@Ab2-NTDNA: Primary antibody specific for PD-L1 (CD274)_Control secondary antibody (not binding to PD-L1) conjugated with Non-Target ssDNA; and Abl_Ab2-TDNA: Primary antibody for PD-L1 (CD274)_Secondary antibody (specific to PD-L1) conjugated with target ssDNA).

[0030] FIG. 3. Analysis of the sensing element (i.e., transfer curve for gFET analysis) showing the response of the chimera probe-conjugated Casl3a-gRNA complex (Casl3a-gRNA) upon addition of 10 pM target miRNA. The binding of the target miRNAs (miR-24, miR-92) to the Casl3a-gRNA complex resulted in a pronounced negative dirac voltage shift compared to the control miRNA (miR-159). The observed shift was significantly greater in the presence of Casl3a-miRNA complex than with Casl3a alone, indicating a successful target recognition and sensor activation at 60 nM Casl3a. The shift in gFET dirac voltage indicates cleavage of the chimera probe from the surface.

[0031] FIG. 4. Transfer curve showing the removal of cleaved chimera probes from the gFET sensor surface following target miRNA detection via Casl3a-mediated collateral cleavage. The activated Casl3a-mediated probe cleavage induced a pronounced positive Dirac voltage shift in test samples (miR-24, miR-92) compared to the control miRNA (miR-159). This shift reflects the dissociation of cleaved probe + bound Casl3a-gRNA-miRNA complex from the graphene surface and was distinct from the negative Dirac shift observed during the miRNA association step.

[0032] FIG. 5. Schematic representation of an integrated CRISPR-Cas-based multi- omics biosensing platform. Whole blood is initially processed through a plasma separation module, after which the plasma is distributed into three analyte-specific pathways. 1) RNA Module: Extracted RNA is reverse transcribed into complementary DNA (cDNA), optionally amplified, and then combined with a target-specific CRISPR-Casl2a and guide RNA complex. 2) DNA Module: Circulating DNA is Attorney Docket No. 15670-0433WO1 (SD2025-050-1) optionally amplified and incubated with Casl2a and a corresponding guide RNA. 3) Protein Module: A pair of DNA-conjugated antibodies binds to a target antigen, forming an antibody-antigen complex. This complex is captured on a DNA- functionalized surface via DNA hybridization. The DNA tag on the secondary antibody is then amplified by PCR and recognized by a target-specific CRISPR- Casl2a complex, leading to its activation. Upon target recognition in each pathway, the activated Casl2a complex triggers collateral cleavage of a chimera reporter probe. This cleavage event generates a measurable optical / el ectrical / electrochemical signal, which is transmitted to a unified signal processing and readout module. The processed data is visualized on a digital interface, enabling simultaneous detection and quantification of DNA, RNA, and protein biomarkers from a single blood sample.

[0033] FIG. 6. Schematic workflow of a multi-omics-based diagnostic platform integrating CRISPR-Casl2a-enabled detection. The system is organized into two main sections: (1) Sample Processing and (2) Detection, Signal Readout, and Analysis. The sample processing unit includes the following modules. Module 1 : plasma separation from whole blood. Module 2: Target-specific analyte isolation (RNA, DNA, and proteins). Module 3: Nucleic acid amplification, where RNA is reverse transcribed into cDNA. Both the target DNA present in the sample and the DNA tags on antibodies bound to the target protein are directly amplified. The detection, signal readout, and analysis pipeline includes the following modules. Module 4: Target recognition is achieved by incubating the amplified DNA derived from RNA (via cDNA), amplified circulating DNA, and primary antibody-protein- secondary antibody complex with DNA tag with CRISPR-Casl2a and guide RNA complexes, leading to Casl2a activation. Module 5: A sensing chamber is equipped with chimera probes that can detect target- induced Casl2a activity. Upon target recognition, Casl2a-mediated collateral cleavage of the chimera probes generates a measurable optical / electrical / electrochemical signal. Module 6: A shared signal readout system captures and transmits the optical / electrical / electrochemical output for downstream processing. Module 7: The data is further refined through a noise filtration module and interpreted using a pre-configured analytical algorithm for clinical decision support.

[0034] FIG. 7. A schematic flow chart illustrating the workflow of the multi-omics sensor assay. 704, 706, and 708 show that each step of contacting (704), measuring Attorney Docket No. 15670-0433WO1 (SD2025-050-1)

[0035] (706), and determining (708) can be performed in separate vessels for each target molecule, where each vessel corresponds to a respective target molecule. 712, 714, and 716 show that each step of contacting (712), measuring (714), and determining (716) can be performed in a common vessel (e.g., the same vessel) containing at least two target molecules or all target molecules.

[0036] DETAILED DESCRIPTION

[0037] Provided herein are methods and materials for simultaneous detection of one or more target analytes in a sample. For example, the present methods and materials relate to a multi-omics assay that can simultaneously detect DNA, RNA, and / or polypeptides in a sample. Compared to traditional methods of profiling nucleic acid molecules (e.g., DNA and RNA) and polypeptides, which are often time-consuming and resource-intensive, the present methods and materials reduce consumable and instrument costs without compromising sensitivity or specificity. For example, the methods and materials provided herein require minimal changes in components to simultaneously profile nucleic acid molecules and polypeptides with high sensitivity and specificity, without the need for amplification.

[0038] Further provided herein are methods and materials for simultaneously determining the presence of one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) target analytes in a sample. For example, the methods and materials include (a) isolating one or more analytes (e.g., DNA, RNA, or polypeptide) from a sample and converting the one or more analytes into one or more target molecules, (b) contacting the one or more target molecules with an activatable Cas construct and a cleavable probe, (c) measuring a signal readout from the Cas- mediated collateral cleavage of the probe, and (d) determining the presence of the one or more target analytes in the sample.

[0039] In some embodiments, the methods and materials described herein include isolating one or more analytes from a sample. For example, an analyte that can be isolated from a sample can be a DNA molecule, an RNA molecule, or a polypeptide. In some embodiments, the sample used to isolate analytes from is a biological sample or an environmental sample. For example, a biological sample can be a tissue (brain tissue, lung tissue, heart tissue, muscle tissue, liver tissue, kidney tissue, adipose tissue, skin tissue, or tumor tissue), a cell (e.g., epithelial cells, muscle cells, nerve cells, blood cells, bone cells, adipocytes, or stem cells), a body fluid (e.g., blood, Attorney Docket No. 15670-0433WO1 (SD2025-050-1) plasma, saliva, urine, sweat, tears, amniotic fluid, synovial fluid, or pleural fluid), or a biopsy sample. In some embodiments, the biological sample is isolated from a mammal described herein. In some embodiments, an environmental sample used to isolate analytes from is soil, water, sediments, ice, biofdms, or waste materials. In some embodiments, the methods provided herein includes sample processing steps. For example, a biological sample such as blood that has been isolated from a mammal can be further processed to separate plasma from blood. The resulting plasma can be used to isolate analytes (e.g., DNA, RNA, and / or polypeptides) within the plasma sample.

[0040] In some embodiments, the analytes are nucleic acid molecules. For example, the nucleic acid molecules can be a DNA molecule (e.g., a genomic DNA, complementary DNA (cDNA), circular DNA, mitochondrial DNA, chloroplast DNA, dsDNA, ssDNA, or linear DNA) or an RNA molecule (e.g., mRNA, tRNA, rRNA, regulatory RNA, microRNA, long non-coding RNA (IncRNA), or circular RNA). In some embodiments, the analytes are polypeptides. In some embodiments, the analytes are DNA, RNA, polypeptides, or any combinations thereof.

[0041] Any appropriate method can be used to isolate analytes from a sample (e.g., plasma or blood). For example, DNA can be isolated from plasma using extraction methods such as microfluidic solid-phase extraction on silica pillars / membranes or on-chip magnetic -bead capture with integrated magnets or other compatible methods. RNA can be isolated from plasma using extraction methods such as on-chip magnetic- bead purification (RNase-free), microfluidic silica surface capture with pH-controlled bind / elute, or electrophoretic separation and preconcentration technique. Polypeptide can be isolated from plasma using protein purification methods such as on-chip immunoaffinity capture (antibody / aptamer-coated beads or surfaces).

[0042] In some embodiments, the methods and materials described herein include converting the one or more analytes (e.g., DNA, RNA, or polypeptides) into one or more target molecules (e.g., DNA molecule, cDNA molecule, or DNA-tagged antibody-polypeptide complex). In some embodiments, a target molecule is a nucleic acid molecule capable of forming complementary base pairs with other nucleic acid molecules. For examples, a target molecule could be a DNA, an RNA, or synthetic analogs thereof. In some cases, the nucleic acid molecule could be single stranded, double stranded, circular, or linear. In some embodiments, the target molecule (e.g., a Attorney Docket No. 15670-0433WO1 (SD2025-050-1)

[0043] DNA) could be amplified using any appropriate method of amplification. In some embodiments, a target molecule is a DNA molecule, a cDNA molecule, and / or a DNA-tagged antibody -polypeptide complex. For instance, RNA molecules can be converted into cDNA molecules via reverse transcription. In some embodiments, polypeptides are contacted with a pair of DNA-conjugated antibodies and bind to the pair of the DNA-conjugated antibodies, and converts the polypeptide into a DNA- tagged antibody polypeptide complex. The DNA-tagged antibody polypeptide complex can include a primary antibody with a DNA tag and a secondary antibody with a DNA tag. In some embodiments, the primary and secondary antibodies are monoclonal antibodies, polyclonal antibodies, or nanobodies. In some embodiments, the antibodies are fully split antibodies, partially split antibodies, or no-split antibodies. Non-limiting examples of fully split antibodies include Fab fragments, F(ab’)2 fragments, or single-chain variable fragments (scFv). Non-limiting examples of partially split antibodies include half-antibodies, heavy chain-only antibodies, or Fc fragments. In some embodiments, the methods and materials provided herein include using the antibody fragments described herein for capture, detection, and / or signal amplification within a lab-on-chip system.

[0044] In some embodiments, the methods and materials described herein include using a DNA-functionalized surface for the simultaneous detection of one or more analytes in a sample. For instance, a DNA-functionalized surface can be used to capture the DNA tag on the primaiy antibody bound to the DNA-tagged antibody polypeptide complex. A DNA-functionalized surface can be a surface (e.g., gold, silicon, glass, or polymer films) that has been chemically modified to immobilize ssDNA, ssRNA, or chimera molecules (e.g., molecules containing DNA and RNA bases in a single strand) on its surface, allowing specific cleavage via activated Cas 12 or Cas 13 complex. In some cases, DNA molecules (e.g., DNA probes) can be attached to the surface via any appropriate techniques (e.g., covalent bonding, adsorption, avidin / streptavidin-biotin interaction) using any appropriate linkers (e.g., thiol linkers, amine linkers, glutaraldehyde linkers, 3-aminopropyltriethoxysilane (APTES) linker, or click chemistry linker). In some embodiments, the DNA- functionalized surface is a graphene field-effect transistor (gFET) sensor. In some embodiments, the gFET surface is modified with 1 -pyrenebutyric acid (PBA), 1 - pyrenebutanoic acid succinimidyl ester (PBASE), pyrene-maleimide derivatives, Attorney Docket No. 15670-0433WO1 (SD2025-050-1) pyrene-His tag, or pyrene amine. In some embodiments, the gFET is activated via EDC / NHS (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / (N- hydroxysuccinimide) chemistry to facilitate peptide bond formation with the DNA molecule (e.g., DNA probe). In some embodiments, when a target molecule (e.g., DNA molecule) binds to the immobilized DNA molecule on the DNA-functionalized surface (e.g., gFET sensor), it induces a detectable change in the current.

[0045] In some embodiments, the target molecules (e.g., DNA molecule, cDNA molecule, or DNA-tagged antibody-polypeptide complex) can be optionally amplified. For example, DNA molecules isolated from the sample or cDNA molecules can be optionally amplified (e.g., via polymerase chain reaction (PCR)). In another example, the DNA tag on the secondary antibody bound to the DNA-tagged antibody polypeptide complex can be optionally amplified (e.g., via polymerase chain reaction (PCR)).

[0046] In some embodiments, the methods and materials described herein include incubating the one or more target molecules (e.g., DNA, cDNA, or DNA-tagged antibody polypeptide complex) with a Cas construct and a cleavable probe (e.g., a chimera probe). A Cas construct can contain a Cas endonuclease that directs DNA and RNA cleavage, a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA) or a guide RNA (gRNA) that direct the Cas endonuclease to a specific target DNA sequence. Any appropriate Cas endonuclease can be used. In some embodiments, the Cas endonuclease is Casl2 (e.g., LbCasl2a) or Casl 3 (e.g., LwaCasl3a), Cas9, CaslO, Cas3, Casl2f, Casl4, Cas (CasPhi), or CasX (Casl2e). Once incubated with the Cas construct, the corresponding guide RNA directs the Cas endonuclease (e.g., Casl2 or Casl3) to recognize and bind the one or more target molecules described herein (e.g., DNA molecules, cDNA molecules, DNA tag on the secondary antibody bound to the DNA-tagged antibody polypeptide complex), which forms a target-specific CRISPR- Cas complex (e.g., target-specific CRISPR- Cas 12a complex) leading to the activation of the Cas construction. The activated Cas complex triggers collateral cleavage of the cleavable probe. The cleavable probe can be a nucleic acid hybrid probe. For instance, a nucleic acid hybrid probe can be a chimera probe, a fragment of a single-stranded DNA or RNA, a double-stranded DNA or RNA, or a partially double-stranded DNA or RNA. In some embodiments, the cleavable probe is an enzyme-cleavable probe, a photolabile-cleavable probe, or a pH- Attorney Docket No. 15670-0433WO1 (SD2025-050-1) cleavable probe. In some cases, the chimera probe includes both DNA and RNA bases within a single strand. In some embodiments, the nucleic acid hybrid probe comprises a donor fluorophore and an acceptor fluorophore. For example, a donor fluorophore can be 6-carboxyfluorescein (6FAM), hexachloro-fluorescein (HEX), tetrachlorofluorescein (TET), 2’,7’-Dimethoxy-4’,5’-dichloro-6-carboxyfluorescein (JOE), alexa fluor 488, or Cy3. A donor fluorophore can be a fluorescent dye attached the 5’ end of the probe. When excited, the donor fluorophore emits light. For example, 6FAM when excited, emits green fluorescence. In some cases, an acceptor fluorophore can be Iowa Black-Hole quencher, any appropriate Black Hole Quenchers, or dabcyl acid. An acceptor agent can be an acceptor or quencher fluorophore attached to the 3 ’ end of the probe. The second agent absorbs the fluorescence emitted by the first agent when the cleavable probe is intact.

[0047] In some embodiments, the methods and materials described herein include measuring a signal readout from the Cas-mediated collateral cleavage of the probe. For example, upon activation by target recognition, Casl2 or Casl3 exhibits collateral cleavage of the cleavable probe, which separates the first agent (e.g., 6FAM) from the second agent (e.g., Iowa Black-Hole quencher), allowing fluorescence emission. The fluorescence emitted is then detected and measured as a signal readout. In some embodiments, the signal is an optical signal, an electrical signal, or an electrochemical signal. In some embodiments, the signal readout produced by the Cas-mediated collateral cleavage of the cleavable probe includes emission of fluorescence, luminescence, absorption spectroscopy, electrochemical redox reaction, gFET sensor output (e.g., dirac point shift, current, gate voltage, or transfer curve), capacitance, resistance, or conductance. In some embodiments, the signal readout includes data refinement through noise fdtration module, pre-configured analytical algorithm, feature-extraction algorithm, classification of positive versus negative signal, or decision threshold module.

[0048] In some embodiments, the methods described herein can include further amplification of the signal. For example, the signal can be amplified by polymerase chain reaction (PCR); increasing time or temperature; adding an additional Cas construct; adding an additional cleavable probe (e.g., chimera probe) to the surface; adding an additional dsDNA (e.g., a target dsDNA), ssDNA (e.g., a target ssDNA), Attorney Docket No. 15670-0433WO1 (SD2025-050-1)

[0049] RNA (e.g., a target RNA), cDNA (e.g., a target cDNA), and / or ssDNA tagged antibodies (e.g., a target ssDNA tagged antibody).

[0050] In some embodiments, the methods and materials provided herein includes determining the presence of one or more target analytes (e.g., one or more target gene of interest) in a sample (e.g., a biological sample). For example, the presence of one or more target analytes can be determined by visualizing the refined and processed data from the signal readout on a digital interface, enabling simultaneous detection and quantification of DNA, RNA, and polypeptides, or any combinations thereof from a single sample. In some embodiments, the processed data will be categorized based on the type of the analyte (e.g., DNA, RNA, or polypeptide), threshold (e.g., concentration thresholds, quantitative ranges), or disease / condition classification. For example, a minimum signal threshold can be calculated based on the signal readout value of a non-target analyte ± 3 standard deviation (SD), where any signal readout value that is equal or below that value (i.e., signal readout value of a non-target analyte ± 3 SD) can be deemed as a negative signal readout, whereas any signal readout value that is greater than that value (i.e., signal readout value of a non-target analyte ± 3 SD) can be deemed as a positive signal readout. In some embodiments, scoring criteria can be used based on drift-corrected endpoint signal (e.g., AF / Fo, Ai, AVDirac), signal to noise ratio vs run-specific noise, specificity index vs non-target panel, and / or replicate coefficient of variance.

[0051] In some embodiments, the target analyte is any gene of interest. In some embodiments, a target analyte is a target gene of interest. For example, the methods and materials provided herein can be used to simultaneously determine the presence of one or more target genes of interest associated with a disease, a disorder, and / or a condition. For instance, the methods and materials provided here can be used to simultaneously determine the presence of one or more target genes of interest associated with a cancer or an infectious disease in any appropriate mammal. Nonlimiting examples of mammals include humans, non-human primates (e.g., monkeys), dogs, cats, pigs, horses, cows, sheep, goats, rabbits, mice, and rats.

[0052] In some embodiments, cancers that can be detected as described herein include solid tumor or blood cancer. Non-limiting examples of solid tumors that can be detected using the methods described herein include head and neck squamous cell carcinoma, pancreatic cancer, renal carcinoma, breast cancer, lung cancer, prostate Attorney Docket No. 15670-0433WO1 (SD2025-050-1) cancer, glioma, melanoma, keratinocyte cancer, ovarian cancer, liver cancer, kidney cancer, bladder cancer, thyroid cancer, sarcoma, stomach cancer, cervical cancer, endometrial cancer, esophageal cancer, thymoma, soft tissue sarcoma, bone cancer, testicular cancer, penile cancer, gallbladder cancer, uterine sarcoma, adrenal gland cancer, ampullary cancer, hepatic angiosarcoma, nasal, or paranasal sinus cancer. Non-limiting examples of blood cancers that can be detected using the methods described herein include leukemia, lymphoma, myeloma, myelodysplastic syndrome, or myeloproliferative neoplasm. In some embodiments, infectious diseases that can be detected as described herein include viral infections, bacterial infections, fungal infections, parasitic infections, or prion infections. Non-limiting examples of viral infections include common cold, influenza, herpes, chickenpox, COVID-19, mumps, human papillomaviral (HPV) infections, rubella, measles, dengue, AIDS, viral gastroenteritis, viral hepatitis, conjunctivitis, Ebola, or zika virus. Non-limiting examples of bacterial infections include gastroenteritis, pseudomonas infection, sexually transmitted infections, bacterial pneumonia, urinary tract infection, tuberculosis, leptospirosis, cholera, or methicillin-resistant staphylococcus aureus (MRSA). Non-limiting examples of fungal infections include ringworm, onychomycosis, candidiasis, sporotrichosis, chromoblastomycosis, eumycetoma, histoplasmosis, coccidioidomycosis, blastomycosis, aspergillosis, Candida urinary tract infection, invasive candidiasis, pneumocystis pneumonia, mucormycosis, or cryptococcosis. Non-limiting examples of parasitic infections malaria, pinworms, toxoplasmosis, giardiasis, trichomoniasis, cryptosporidiosis, cyclosporiasis, cysticercosis, strongyloidiasis, tapeworm infections, chagas disease, leishmaniasis, or schistosomiasis. Non-limiting examples of prion infections include Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, chronic wasting disease (CWD), transmissible mink encephalopathy (TME), or feline spongiform encephalopathy.

[0053] In some embodiments, each step of contacting (FIG. 7, 704), measuring (FIG. 7, 706), and determining (FIG. 7, 708) is performed in separate vessels containing aliquots pooled from one or more target molecules (e.g., DNA molecule, cDNA molecule, or DNA-tagged antibody-polypeptide complex), where each vessel corresponds to a respective target molecule (e.g., DNA, RNA, or polypeptide). A Attorney Docket No. 15670-0433WO1 (SD2025-050-1) vessel can be any appropriate vessel. For example, a vessel can be a flask, a plate, a container, a vial, a petri dish, a dish, or a well.

[0054] In some embodiments, the methods described herein can include performing the step of contacting one or more target molecules (e.g., DNA molecule, cDNA molecule, or DNA-tagged antibody-polypeptide complex) with an activatable Cas construct and a cleavable probe in separate vessels for each target molecule (FIG. 7, 704). For example, for each analyte (e.g., DNA, RNA, and polypeptide) converted into a target molecule, the step of contacting the target molecules with an activatable Cas construct and a cleavable probe can be performed in separate vessel.

[0055] In some embodiments, the methods described herein can include performing the step of measuring the signal readout from a Cas-mediated collateral cleavage of the probe in separate vessels for each target molecule (e.g., DNA, RNA, and polypeptide) (FIG. 7, 706). For example, for each analyte converted into a target molecule (e.g., DNA molecule, cDNA molecule, or DNA-tagged antibody- polypeptide complex), the methods described herein can include performing the step of measuring the signal readout from the Cas-mediated collateral cleavage of the cleavable probe in separate vessels. In some embodiments, the methods described herein includes measuring the signal readout using a distinct signal for each target molecule. For example, for each analyte, the signal readout from the Cas-mediated collateral cleavage of the cleavable probe can be measured using separate signals. In some embodiments, the signal readout for each target molecule is a distinct signal. For example, the signal readout for DNA is different from that of RNA and / or that of DNA-tagged antibody-polypeptide complex.

[0056] In some embodiments, the methods described herein can include performing the step of determining the presence of one or more target analytes (e.g., a target gene of interest) in separate vessels for each target analyte (FIG. 7, 708). For example, the methods described herein can include performing the step of determining the presence of one or more target genes of interest associated with a disease, a disorder, and / or a condition in the sample in separate vessels.

[0057] In some embodiments, each step of contacting (FIG. 7, 712), measuring (FIG. 7, 714), and determining (FIG. 7, 716) is performed in a common vessel (e.g., the same vessel) containing aliquots pooled from one or more target molecules (e.g., DNA molecule, cDNA molecule, or DNA-tagged antibody-polypeptide complex). In Attorney Docket No. 15670-0433WO1 (SD2025-050-1) some embodiments, each step of contacting, measuring, and determining is performed in a common vessel (e.g., the same vessel) containing aliquots pooled from at least two target molecules (e.g., DNA molecule, cDNA molecule, DNA-tagged antibody- polypeptide complex, or any combinations thereof). The selection between performing a given step in a common vessel for one or more target molecules or in separate vessels for each target molecule can be made independently from each other, providing a flexibility in the workflow. For example, each step of contacting and measuring can be performed in separate vessels for each target molecule, while the step of determining can be performed in a common vessel for all the target molecules (FIG. 7, 704, 706 and 716). In another example, the step of contacting can be performed in separate vessels for each target molecule while each step of measuring and determining can be performed in a common vessel for all the target molecules (FIG. 7, 704, 714 and 716).

[0058] In some embodiments, the methods described herein can include performing the step of contacting one or more target molecules (e.g., DNA molecule, cDNA molecule, or DNA-tagged antibody-polypeptide complex) with an activatable Cas construct and a cleavable probe in the same vessel (FIG. 7, 712). For example, DNA molecule, cDNA molecule, and DNA-tagged antibody-polypeptide complex can be combined together in one vessel to perform the step of contacting these target molecules with an activatable Cas construct and a cleavable probe. In some embodiments, the methods described herein can include performing the step of contacting one or more target molecules with an activatable Cas construct and a cleavable probe in the same vessel for at least two target molecules (e.g., DNA molecule, a cDNA molecule, or a DNA-tagged antibody-polypeptide complex, or any combinations thereof). For example, DNA and cDNA can be combined together in one vessel to perform the step of contacting the target molecules with an activatable Cas construct and a cleavable probe, while the same step can be performed in a separate well for DNA-tagged antibody-polypeptide complex.

[0059] In some embodiments, the methods described herein can include performing the step of measuring the signal readout from the Cas-mediated collateral cleavage of the cleavable probe in the same vessel for all the target analytes (FIG. 7, 714). For example, DNA molecule, cDNA molecule, and DNA-tagged antibody-polypeptide complex can be combined together in one vessel to perform the step of measuring the Attorney Docket No. 15670-0433WO1 (SD2025-050-1) signal readout from the Cas-mediated collateral cleavage of the cleavable. In some embodiments, the methods described herein can include performing the step of measuring the signal readout from the Cas-mediated collateral cleavage of the cleavable probe in the same vessel for at least two target molecules (e.g., DNA molecule, cDNA molecule, or DNA-tagged antibody-polypeptide complex, or any combinations thereof)- For example, DNA and cDNA can be combined together in one vessel to perform the step of measuring the signal readout from the Cas-mediated collateral cleavage of the cleavable probe, while the same step can be performed in a separate well for DNA-tagged antibody-polypeptide complex. In some embodiments, the signal readout for all the target analytes is the same signal. For example, the signal readout for DNA, RNA, and DNA-tagged antibody-polypeptide complex can be a fluorescence signal. In some embodiments, the signal readout for at least two target analytes is the same signal. For example, the signal readout for DNA and RNA can be a fluorescence signal, while the signal readout for DNA-tagged antibody-polypeptide complex can be a gFET sensor output.

[0060] In some embodiments, the methods described herein can include performing the step of determining the presence of all the target analytes (e.g., a target gene of interest) in the same vessel (FIG. 7, 716). For example, the methods described herein can include performing the step of determining the presence of one or more target genes of interest associated with a disease, a disorder, and / or a condition in the same vessel. In some embodiments, the step of determining the presence of target analytes is determined in the same vessel for at least two target analytes. For example, the presence of DNA and RNA can be determined in the same vessel, while the presence of DNA-tagged antibody-polypeptide complex can be determined in a separate vessel.

[0061] EXAMPLES

[0062] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0063] Example 1: Multi-Omics Assay Method

[0064] After plasma separation, the sample stream is split into three modules: RNA, DNA, and protein (FIGS. 5, 6, and 7). RNA and DNA modules contain freeze-dried nuclease inhibitors (RNase inhibitor in the RNA lane; DNase inhibitor in the DNA lane) together with a protease (e.g., Proteinase K) to remove residual proteins. The Attorney Docket No. 15670-0433WO1 (SD2025-050-1) outflow from each module enters a short protease-quench chamber (protease inhibitor) to prevent any carryover activity that could interfere with the downstream Casl2a reaction. The quenched sample is divided into two amplification chambers preloaded with specific primers and polymerases plus on-chip heating. The DNA lane amplifies genomic / cell-free DNA and generates ssDNA input. The RNA lane performs reverse transcription and amplification and yields single stranded cDNA. The resulting products are mixed with Casl2a RNPs, and the recognition of the target activated Casl2a, which cleaves the chimera reporter probe, producing an optical / electrical / electrochemical signal. In the protein module, plasma is routed to an antibody -binding module where a primary / secondary antibody pair forms a sandwich in the presence of the target. The complex is captured on a surface bearing a ssDNA capture probe (e.g., via DNA-tagged antibodies / proximity ligation). After washing away the excess sample, the captured complex primes DNA polymerization, generating an amplified DNA barcode that is subsequently recognized by Casl2a, which cleaves the same chimera reporter probe. The target-dependent probe cleavage yields the corresponding signal readout (e.g., optical / electrical / electrochemical readout).

[0065] Example 2: Detection of target genes using CRISPR-Casl2 and CRISPR-Casl3 systems

[0066] As a proof-of-concept, a fluorescence-based detection assay was performed using Casl2 with both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) BRAF gene targets. A unique chimera probe was designed that contained both DNA and RNA bases within a single strand, allowing detection of both types of nucleic acids using the same probe. This probe incorporates a 5’-end 6FAM fluorophore and a 3’ Iowa Black-Hole quencher. Upon excitation at 480 nm, emission was measured and recorded at 520 nm (FIG. 1A). The results showed successful detection of both dsDNA (FIG. 2A) and ssDNA (FIG. 2B) targets. Notably, Casl2a was able to detect ssDNA even in the absence of a protospacer adjacent motif (PAM), expanding its utility for sequence-agnostic genome detection.

[0067] In parallel, LwaCasl3a was used with specific guide RNAs targeting five different microRNA: miR7a, miR21, miR23, miR24, and miR92 (Table 1). All target Attorney Docket No. 15670-0433WO1 (SD2025-050-1) miRNAs were successfully detected compared to the non-target control (miRNA 159) (FIG. 2E).

[0068] Table 1. Sequence of miRNA tested using Casl3a fluorescence assay and gFET assay

[0069] For antigen / protein detection, a Casl2a-based system and antibody pairs were used. To enhance signal amplification, a DNA / RNA chimera probe was used, and a newly designed ssDNA target containing multiple Casl2a binding sites for a secondary antibody was introduced (FIG. 1 C). The primary antibody was immobilized on a 96-well plate using EDC / NHS chemistry, and a PD-L1 protein (a biomarker for non-small cell lung cancer) served as a target antigen. In the presence of PD-L1, the ssDNA conjugated with the secondary antibody enabled Casl2a- mediated cleavage of the chimera probe and fluorescence enhancement via collateral activity, confirming the detection of PD-L1 (FIG. 2D).

[0070] The integrated CRISPR-based detection system was validated on a graphene field-effect transistor (gFET) platform. Using LwaCasl3a, the synthetic miRNA targets and the corresponding crRNAs were tested (FIG. IB). The sensor response exhibited a concentration-dependent shift in the charge neutrality point (Dirac point) of the probe-conjugated graphene surface. The 5’ end of the probe was labeled with 6FAM, enabling charge interaction with graphene, while the 3 ’ end of the probe was amine-functionalized for covalent conjugation (FIG. IB). The graphene surface was modified with 1 -pyrenebutyric acid (PBA) and activated via EDC / NHS chemistry to facilitate peptide bond formation with the probe. Upon the introduction of femtomolar to nanomolar concentrations of target miRNA, Casl3a activation led to collateral cleavage of the probe. This cleavage caused the release of 6FAM from the surface, Attorney Docket No. 15670-0433WO1 (SD2025-050-1) thereby altering the Dirac potential of the gFET sensor (FIGs. 3A-3F, association signal; FIGs. 4A-4F, dissociation signal), validating the detection mechanism.

[0071] OTHER EMBODIMENTS

[0072] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 15670-0433 WO 1 (SD2025-050-1)WHAT IS CLAIMED IS:1 . A multi-omics method for simultaneously determining the presence of one or more target analytes in a sample, the method comprising:(a) isolating one or more analytes from the sample and converting the one or more analytes into one or more target molecules,(b) contacting the one or more target molecules with an activatable Cas construct and a cleavable probe,(c) measuring a signal readout from the Cas-mediated collateral cleavage of the cleavable probe, and(d) determining the presence of the one or more target analytes in the sample.

2. The method of claim 1, wherein the sample is a biological sample or an environmental sample.

3. The method of claim 2, wherein the biological sample comprises a tissue, a cell, a body fluid, or a biopsy sample.

4. The method of claim 1, wherein the environmental sample comprises soil, water, sediments, ice. biofilms, waste materials, or rocks.

5. The method of any one of claims 1-3, wherein the biological sample is blood.

6. The method of any one of claims 1-5, further comprising processing the sample.

7. The method of claim 6. wherein processing the sample comprises separating plasma from blood.

8. The method of claim 1, wherein the one or more analytes comprise a DNA molecule, an RNA molecule, and / or a polypeptide.Attorney Docket No. 15670-0433 WO 1 (SD2025-050-1)9. The method of any one of claims 1-8, wherein converting one or more analytes comprises reverse transcribing an RNA molecule into a cDNA molecule.

10. The method of any one of claims 1-8, wherein converting one or more analytes comprises contacting the polypeptide with pair of DNA-conjugated antibodies forming a DNA-tagged antibody -polypeptide complex.

11. The method of claim 10, wherein the pair of DNA-conjugated antibodies comprises a primary antibody with a DNA tag and a secondary antibody with a DNA tag forming the DNA-tagged antibody-polypeptide complex.

12. The method of any one of claims 1-11, wherein the antibody comprises a monoclonal antibody, a polyclonal antibody, or a nanobody.

13. The method of any one of claims 1-11, wherein the antibody comprises a fully split antibody, a partially split antibody, or a no-split antibody.

14. The method of claim 13, wherein the fully split antibody comprises a Fab fragment, an F(ab’)2 fragments, or a single-chain variable fragment (scFv).

15. The method of claim 13, wherein the partially split antibody comprises a halfantibody, a heavy chain-only antibody, or an Fc fragment.

16. The method of any one of claims 1-15, wherein the DNA tag on the primary antibody bound to the DNA-tagged antibody-polypeptide complex is captured on a DNA-functionalized surface.

17. The method of any one of claims 1-16. wherein the one or more target molecules comprise a cDNA molecule, a DNA molecule, or the DNA-tagged antibody-polypeptide complex.Attorney Docket No. 15670-0433 WO 1 (SD2025-050-1)18. The method of claim 1. wherein the cleavable probe is a nucleic acid hybrid probe.

19. The method of claim 18, wherein the nucleic acid hybrid probe comprises a chimera probe, a fragment of a single-stranded DNA or RNA, a doublestranded DNA or RNA, or a partially double-stranded DNA or RNA.

20. The method of any one of claims 1-18, wherein the cleavable probe comprises an enzyme-cleavable probe, a photolabile-cleavable probe, or a pH-cleavable probe.

21. The method of claim 19, wherein the chimera probe comprises both DNA and RNA bases within a single strand.

22. The method of claim 19, wherein the nucleic acid hybrid probe comprises a donor fluorophore and an acceptor fluorophore.

23. The method of claim 1, wherein the Cas construct comprises a guide RNA and Cas 12 and / or Cas 13.

24. The method of any one of claims 1-23, wherein contacting comprises binding of the Cas construct to the one or more target molecule.

25. The method of any one of claims 1-23. wherein contacting comprises binding of the Cas construct to the DNA tag on the secondary antibody bound to the DNA-tagged antibody-polypeptide complex.

26. The method of claim 1, wherein the signal comprises an optical signal, an electrical signal, an electrochemical signal.

27. The method of claim 1, wherein the signal readout comprises fluorescence, luminescence, absorption spectroscopy, electrochemical redox reaction,Attorney Docket No. 15670-0433 WO 1 (SD2025-050-1) graphene field-effect transistor (gFET) sensor output, capacitance, resistance, or conductance.

28. The method of claim 1, further comprising amplifying the signal by polymerase chain reaction (PCR); increasing time or temperature; adding an additional Cas construct; adding an additional cleavable chimera probe; adding an additional dsDNA, ssDNA, RNA, cDNA, and / or ssDNA tagged antibody.

29. The method of claim 1, wherein measuring the signal readout comprises data refinement through noise filtration module, pre-configured analytical algorithm, feature-extraction algorithm, classification of positive versus negative signal, or decision threshold module.

30. The method of claim 1, wherein the presence of one or more target analytes in a sample indicates a cancer or an infectious disease.

31. The method of claim 1 , wherein contacting the one or more target molecules with the activatable Cas construct and the cleavable probe is performed in separate vessels for each target molecule.

32. The method of claim 1, wherein contacting the one or more target molecules with the activatable Cas construct and the cleavable probe is performed in the same vessel for the one or more target molecules.

33. The method of claim 1, wherein contacting the one or more target molecules with the activatable Cas construct and the cleavable probe is performed in the same vessel for at least two target molecules.

34. The method of claim 1, wherein measuring the signal readout from the Cas- mediated collateral cleavage of the probe is performed in separate vessels for each target molecule.Attorney Docket No. 15670-0433 WO 1 (SD2025-050-1)35. The method of claim 1. wherein measuring the signal readout from the Cas- mediated collateral cleavage of the probe is performed in the same vessel for the one or more target molecules.

36. The method of claim 1, wherein measuring the signal readout from the Cas- mediated collateral cleavage of the probe is performed in the same vessel for at least two target molecules.

37. The method of claim 1, wherein the signal readout is measured using the same signal for the one or more target molecules.

38. The method of claim 1, wherein the signal readout is measured using a distinct signal for each target molecule.

39. The method of claim I . wherein the signal readout is measured using the same signal for at least two target molecules.

40. The method of claim 1, wherein the presence of the one or more target analytes in the sample is determined in the same vessel.41 . The method of claim 1 , wherein the presence of the one or more target analytes in the sample is determined in separate vessels for each target analyte.

42. The method of claim 1, wherein the presence of the one or more target analytes in the sample is determined in the same vessel for at least two target analytes.

43. The method of claim 30, wherein the cancer comprises a solid tumor or a blood cancer.

44. The method of claim 43, wherein the solid tumor comprises head and neck squamous cell carcinoma, pancreatic cancer, renal carcinoma, breast cancer, lung cancer, prostate cancer, glioma, melanoma, keratinocyte cancer, ovarianAttorney Docket No. 15670-0433 WO 1 (SD2025-050-1) cancer, liver cancer, kidney cancer, bladder cancer, thyroid cancer, sarcoma, stomach cancer, cervical cancer, endometrial cancer, esophageal cancer, thymoma, soft tissue sarcoma, bone cancer, testicular cancer, penile cancer, gallbladder cancer, uterine sarcoma, adrenal gland cancer, ampullary cancer, hepatic angiosarcoma, nasal, or paranasal sinus cancer.

45. The method of claim 43, wherein the blood cancer is leukemia, lymphoma, myeloma, myelodysplastic syndrome, or myeloproliferative neoplasm.

46. The method of claim 30, wherein the infectious disease comprises a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a prion infection.

47. The method of any one of claims 2-46, wherein the biological sample is obtained from a subject, wherein the subject is a mammal.

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