Devices, systems and methods for analysis of nucleic acids

Abstract

Various compositions and diagnostic devices for the detection and measurement of nucleic acids are provided. Also provided are systems comprising the same, and methods of using the same. In some embodiments, compositions and devices comprise or are configured to be used in combination with one or more of a guide nucleic acid, a programmable nuclease, and reporters. In some embodiments, presence, absence, or amount of at least one target nucleic acid is determined by detecting a signal produced upon cleavage of the reporters.

Description

Docket No.203477-779601 DEVICES, SYSTEMS AND METHODS FOR ANALYSIS OF NUCLEIC ACIDS CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 579,479 filed August 29, 2023, which is incorporated herein in its entirety for all purposes. BACKGROUND

[0002] Detection of ailments, especially at the early stages of disease or infection, can provide guidance on treatment or intervention to reduce the progression or transmission of said ailments. Such ailments can be detected at the point of need by devices capable of running diagnostic assays. Various biological species associated with an organism, disease state, phenotype, or genotype can be detected by these devices. Diagnostic systems that detect the presence or absence of nucleic acids often have an advantage in sensitivity and specificity over other test methods, such as those based on antigen detection of the pathogenic organism. In some cases, while antibody / antigen tests may have the ability to directly assay a subject’s immune response to a pathogenic organism, these systems can have sensitivity and specificity challenges related to the response time and the variabilities of antibodies / antigens produced by the subject’s body. However, nucleic acid-based detection systems are not without limitations to detection sensitivity. SUMMARY

[0003] In certain aspects, described herein are embodiments of a system for measuring an amount of a target nucleic acid in a sample. In some embodiments, the system includes a plurality of reaction mixtures, wherein: (a) each reaction mixture of the plurality of reaction mixtures includes a programmable nuclease, a guide nucleic acid, and a reporter; (b) the guide nucleic acid includes a sequence configured to bind to the target nucleic acid; (c) the programmable nuclease is effective to cleave the reporter in response to formation of a complex including the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (d) cleavage of the reporter is effective to produce a detectable product at a rate proportional to the amount of the target nucleic acid present in the sample; (e) the plurality of reaction mixtures includes control reaction mixtures including a predetermined amount of the target nucleic acid; (f) the predetermined amount of target nucleic acid in a first of the control reaction mixtures is at least 10-fold higher than the predetermined amount of target nucleic acid in a second of theDocket No.203477-779601 control reaction mixtures; and (g) the plurality of reaction mixtures includes a sample reaction mixture including a nucleic acid sample to be measured.

[0004] In some embodiments, the control reaction mixtures include three or more control reaction mixtures, wherein each of the three or more control reaction mixtures includes a different predetermined amount of the target nucleic acid. In some embodiments, the predetermined amount of target nucleic acid in the first of the control reaction mixtures is 10,000-fold higher than the predetermined amount of target nucleic acid in the second of the control reaction mixtures. In some embodiments, each reaction mixture of the plurality of reaction mixtures further includes reagents for amplifying the target nucleic acid. In some embodiments, each reaction mixture of the plurality of reaction mixtures further includes reagents for reverse transcribing the target nucleic acid. In some embodiments, the plurality of reaction mixtures are contained in a microfluidic device. In some embodiments, the microfluidic device includes a sample interface configured to receive a sample, and reaction chambers in fluid communication with the sample interface, wherein each reaction chamber includes one of the reaction mixtures of the plurality of reaction mixtures. In some embodiments, the microfluidic device includes an input chamber fluidically connected between the sample interface and the reaction chambers, optionally wherein the sample interface, input chamber, and reaction chambers are fluidically connected by one or more valves. In some embodiments, the input chamber includes lysis reagents. In some embodiments, the system further includes one or more lateral flow assay strips. In some embodiments, the programmable nuclease is a Cas protein, optionally wherein the Cas protein is a Cas12 protein, a Cas13 protein, a Cas14 protein, or a CasPhi protein. In some embodiments, the reporter is coupled to the guide nucleic acid. In some embodiments, the reporter includes a fluorescent label and a quencher.

[0005] In certain aspects, described herein are embodiments of a method for measuring an amount of a target nucleic acid in a sample. In some embodiments, the method utilizes a system disclosed herein. In some embodiments, the method includes: (a) initiating detection reactions by heating the plurality of reaction mixtures to a target temperature; (b) measuring the detectable product at each of a plurality of times for each of the plurality of reaction mixtures; (c) determining a time to result (TTR) for each of the plurality of reaction mixtures, wherein the TTR is a reaction time to reach a predetermined state; (d) comparing the TTR for the sample reaction mixture to the TTRs of the control reaction mixtures; and (e) determining an amount of the target nucleic acid in the sample reaction mixture based on the comparing.

[0006] In some embodiments, the TTR is a time from the initiating the detection reactions to reach a predetermined rate of increase in formation of the detectable product. In some embodiments, the TTR is a time from the initiating the detection reactions to reach aDocket No.203477-779601 predetermined percent of a maximum level of detectable product formed at a last of the plurality of times. In some embodiments, the predetermined percent is at least 20%, 25%, or 30% of the maximum level. In some embodiments, the method further includes using the TTR to generate a standard curve for measuring the target nucleic acid. In some embodiments, the standard curve is about linear on a log scale over a range of amounts of the target nucleic acid per reaction mixture, wherein the range of amounts span at least 2 logs, 3 logs, 4 logs, 5 logs, 6 logs, 7 logs, or 8 logs. In some embodiments, the standard curve is about linear on a log scale over a range of amounts of the target nucleic acid per reaction mixture, wherein the range of amounts includes 1000, 100, 50, 25, or fewer copies of the target nucleic acid. In some embodiments, the standard curve that is about linear on a log scale has an R2value of at least 0.85, 0.9, or 0.95 over the range of amounts. In some embodiments, the TTRs for an amount within the range of amounts is about 1-60 minutes, 1-30 minutes, 1-20 minutes, 1-10 minutes, or 1-5 minutes. In some embodiments, the detection reactions include amplification reactions to amplify the target nucleic acid. In some embodiments, the amplification reactions include loop mediated amplification (LAMP). In some embodiments, the detection reactions include reverse transcription reactions. In some embodiments, the detection reactions include cleaving the reporter in response to formation of the complex including the programmable nuclease, the guide nucleic acid, and the target nucleic acid. In some embodiments, (i) the reporter includes a fluorescent label and a quencher; and (ii) measuring the detectable product includes measuring a level of fluorescence in the reaction mixtures. INCORPORATION BY REFERENCE

[0007] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:Docket No.203477-779601

[0009] FIG.1 shows a schematic of an exemplary workflow for a multiplexed programmable nuclease assay. The illustrated reactions may include detection in the respective reaction chamber, or optionally moved to another location for separate detection reactions.

[0010] FIGS.2A-2B show exemplary results from the generation of an RT-LAMP amplification product (top) from various starting concentrations (0 copies (0cp / rxn), 50 copies (50cp / rxn), 100 copies (100cp / rxn), 500 copies (500cp / rxn), 1000 copies (1000cp / rxn), 5000 copies (5000cp / rxn), or 10000 copies (10000cp / rxn)) of a Respiratory Syncytial Virus (RSV) B target ribonucleic acid and concurrent detection of the amplified target nucleic acids (bottom) in RT-LAMP-DETECTR one-pot assays. Amplification was monitored via generation of a SYTO9 fluorescent signal. Effector protein-based detection was monitored via generation of an Alexa594 fluorescent signal.

[0011] FIG.3 shows a standard curve generated using the TTR data from FIGS.2A-2B.

[0012] FIG.4 shows^the effect of various glycerol concentrations (0% control, 1%, and 5% glycerol) on standard curve generation. Illustrated lines from top to bottom in the graph correspond to the glycerol concentrations from bottom to top in the legend.

[0013] FIG.5 shows^the effect of various sucrose concentrations (0% control, 1%, 5%, and 10% sucrose) on standard curve generation. Illustrated lines from top to bottom in the graph correspond to the sucrose concentrations from bottom to top in the legend.

[0014] FIG.6 depicts an exemplary high-level workflow for using a detection system, in accordance with some embodiments. The illustrated process provides a method for detecting target nucleic acids from one or more of influenza virus A (IVA), influenza virus B (IVB), respiratory syncytial virus (RSV), SARS-CoV-2 (SC2), and RNase P (RP). The illustrated reaction containers represent reaction locations within a multi-well plate or microfluidic device, which in addition to reaction locations for sample nucleic acids, may include reaction locations for control reactions for each of the one or more targets.

[0015] FIGS.7A-7C illustrates immobilization strategies for CRISPR-Cas diagnostic assay components, as described herein.

[0016] FIG.8 illustrates an embodiment where immobilization strategies are combined to enable CRISPR diagnostic readouts, as described herein.

[0017] FIG.9 presents an overview of sRCA.

[0018] FIG.10 presents an overview of RCA positive feedback for Cas13.

[0019] FIG.11 presents an overview of CasPin.

[0020] FIG.12 presents potential hairpin structures for CasPin.

[0021] FIG.13 presents results for an initial design using two hairpins.

[0022] FIG.14 shows illustrative results for programmable nuclease assays with varyingDocket No.203477-779601 amounts of Bst 2.0 DNA polymerase, in accordance with some embodiments. Bst concentrations in the legend, and corresponding R2-values and plots are correspondingly numbered. Illustrated elements include numbered data in graph corresponding to numbered labels in legend. Labels for lines (1) and (2) in the graph are grouped due to proximity.

[0023] FIG.15 shows illustrative results for programmable nuclease assays with varying amounts of dNTP, in accordance with some embodiments. Illustrated lines from left to right in the top graphs correspond to log concentrations indicated in the legend from bottom to top (with the plot for concentration of zero being the bottom horizontal line).

[0024] FIG.16 shows illustrative results for programmable nuclease assays performed at varying reaction temperatures, in accordance with some embodiments. Illustrated lines from left to right in the graph correspond to the concentrations indicated in the legend from bottom to top (with the plot for concentration of zero being the bottom horizontal line).

[0025] FIG.17 shows illustrative results for programmable nuclease assays with or without the addition of tRNA, in accordance with some embodiments. Illustrated lines from left to right in the top graphs correspond to the concentrations indicated in the legend from bottom to top (with the plot for concentration of zero being the bottom horizontal line). Illustrated vertical groups of data points from left to right in the bottom graphs correspond to log dilutions indicated in the legend from top to bottom (1 to 7).

[0026] FIG.18 shows illustrative results for LAMP amplification (top) and DETECTR (bottom) curves generated for varying amounts of target nucleic acid, and using one of two different Mg2+ / dNTP conditions, in accordance with some embodiments. Reactions in buffer with 1.2mM dNTPs and 7mM MgAOc (designated “1.2 / 7” and labeled “(1)”) are grouped together (at left in each pair). Reaction in buffer with 1.4mM dNTP and 8mM MgAOc (designated “1.4 / 8” and labeled “(2)”) are also grouped together (at right in each pair). DETAILED DESCRIPTION

[0027] The present disclosure provides devices, systems, and methods for measuring an amount of a target nucleic acid in a sample. The systems and methods of the present disclosure can be implemented using devices that are configured for programmable nuclease-based detection. In some embodiments, the devices can be configured for single reaction detection. In some embodiments, the devices can be disposable devices. The devices disclosed herein can be particularly well suited for carrying out highly efficient, rapid, and accurate reactions for detecting whether and how much of a target is present in a sample. The target nucleic acid can comprise a target sequence. As used herein, a target can be referred to interchangeably as a targetDocket No.203477-779601 nucleic acid. Further, a target can be referred to as a target amplicon or a target nucleic acid amplicon if such target undergoes amplification (e.g., through a thermocycling or isothermal amplification process as described elsewhere herein). The target nucleic acid can be a portion of a nucleic acid of interest, e.g., a target nucleic acid from any plant, animal, virus, or microbe of interest. The devices provided herein can be used to perform rapid tests in a single integrated system.

[0028] The target nucleic acid can be a nucleic acid or a portion of a nucleic acid from a pathogen, virus, bacterium, fungi, protozoa, worm, or other agent(s) or organism(s) responsible for and / or related to a disease or condition in living organisms (e.g., humans, animals, plants, crops, and the like). The target nucleic acid can be a nucleic acid, or a portion thereof. The target nucleic acid can be a portion of a nucleic acid from a gene expressed in a cancer or genetic disorder in the sample. The target nucleic acid can be a portion of an RNA or DNA from any organism in the sample. In some embodiments, one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule). A programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter. The programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter. Such a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein can be activated by a target RNA or a target DNA. For example, a programmable nuclease, e.g., a Cas enzyme, can be activated by a target RNA nucleic acid or a target DNA nucleic acid to cleave RNA reporters. In some embodiments, the Cas enzyme can bind to a target ssDNA which initiates trans cleavage of RNA reporters. In some instances, a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA-activated programmable DNA nuclease.

[0029] The nucleic acids described and referred to herein can comprise a plurality of base pairs. A base pair can be a biological unit comprising two nucleobases bound to each other by hydrogen bonds. Nucleobases can comprise adenine, guanine, cytosine, thymine, and / or uracil. In some cases, the nucleic acids described and referred to herein can comprise different base pairs. In some cases, the nucleic acids described and referred to herein can comprise one or more modified base pairs. The one or more modified base pairs can be produced when one or more base pairs undergo a chemical modification leading to new bases. The one or more modified base pairs can be, for example, Hypoxanthine, Inosine, Xanthine, Xanthosine, 7-Methylguanine, 7-Docket No.203477-779601 Methylguanosine, 5,6-Dihydrouracil, Dihydrouridine, 5-Methylcytosine, 5-Methylcytidine, 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC).

[0030] The programmable nuclease can become activated after binding of a guide nucleic acid that is complexed with the programmable nuclease with a target nucleic acid, and the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the target nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal. The reporter and / or the detection moiety can be immobilized on a support medium. Often the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid. In some embodiments, the detectable signal is visualized to assess the presence or concentration of one or more target nucleic acids associated with an ailment, such as a disease, cancer, or genetic disorder.

[0031] The systems and methods of the present disclosure can be implemented using a device that is compatible with any type of programmable nuclease that is human-engineered or naturally occurring. The programmable nuclease can comprise a nuclease that is capable of being activated when complexed with a guide nucleic acid and a target nucleic acid segment or a portion thereof. A programmable nuclease can become activated when complexed with a guide nucleic acid and a target sequence of a target gene of interest. The programmable nuclease can be activated upon binding of a guide nucleic acid to a target nucleic acid and can exhibit or enable trans cleavage activity once activated. In any instances or embodiments where a CRISPR- based programmable nuclease is described or used, it is recognized herein that any other type of programmable nuclease can be used in addition to or in substitution of such CRISPR-based programmable nuclease.

[0032] The systems and methods of the present disclosure can be implemented using a device that is compatible with a plurality of programmable nucleases. The device can comprise a plurality of programmable nuclease probes comprising the plurality of programmable nucleases and one or more corresponding guide nucleic acids. The plurality of programmable nuclease probes can be the same. Alternatively, the plurality of programmable nuclease probes can be different. For example, the plurality of programmable nuclease probes can comprise different programmable nucleases and / or different guide nucleic acids associated with the programmable nucleases.Docket No.203477-779601 Quantification of nucleic acid targets using single reaction assays

[0033] Disclosed herein are systems and methods for quantifying nucleic acid targets using single reaction programmable nuclease-based assays. In some in vitro diagnostics applications, levels of target DNA or RNA, such as viral load, gene expression, or copy number, may be quantified to provide information for clinical decisions. Dose-dependent assay times may be used for quantifying nucleic acids. Disclosed herein are methods of combining the specificity of a programmable nuclease-based detection assay (e.g., DETECTR) with isothermal amplification to quantify target nucleic acids in a sample.

[0034] In some aspects, described herein is a system for measuring an amount of a target nucleic acid in a sample. In some embodiments, the system comprises a plurality of reaction mixtures. In some embodiments, each reaction mixture of the plurality of reaction mixtures comprises a programmable nuclease, a guide nucleic acid, and a reporter. In some embodiments, the programmable nuclease is a Cas protein, such as a Cas12 protein, a Cas13 protein, a Cas14 protein, or a CasPhi protein. In some embodiments, the guide nucleic acid comprises a sequence configured to bind to the target nucleic acid. In some embodiments, the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid. In some embodiments, the reporter is coupled to the guide nucleic acid. In some embodiments, the reporter comprises a fluorescent label and a quencher. In some embodiments, cleavage of the reporter is effective to produce a detectable product at a rate proportional to the amount of the target nucleic acid present in the sample. Additional examples of programmable nucleases, guide nucleic acids, and reporters are described herein.

[0035] In some embodiments, the plurality of reaction mixtures comprises control reaction mixtures comprising a predetermined amount of the target nucleic acid. For example, the control reaction mixtures may comprise three or more control reaction mixtures, wherein each of the three or more control reaction mixtures comprises a different predetermined amount of the target nucleic acid. For example, the predetermined amount of target nucleic acid in a first of the control reaction mixtures may be at least 10-fold or 10,000-fold higher than the predetermined amount of target nucleic acid in a second of the control reaction mixtures. In some embodiments, the plurality of reaction mixtures comprises a sample reaction mixture comprising a nucleic acid sample to be measured. By measuring the signal from reactions with predetermined amounts of target nucleic acid at defined point(s) in a reaction, a standard curve can be generated and used to determine the amount of nucleic acid in a test sample by comparing a signal of the test sample at a corresponding point in the reaction to the standard curve. In someDocket No.203477-779601 embodiments, each reaction mixture of the plurality of reaction mixtures further comprises one or more of: reagents for amplifying the target nucleic acid, and reagents for reverse transcribing the target nucleic acid.

[0036] In some embodiments, the plurality of reaction mixtures is contained in plurality of reaction wells or chambers, e.g., a plurality of wells of a microwell plate, wherein each reaction well or chamber comprises one of the reaction mixtures of the plurality of reaction mixtures.

[0037] In some embodiments, the plurality of reaction mixtures is contained in a microfluidic device, which may comprise: a sample interface configured to receive a sample; reaction chambers in fluid communication with the sample interface, wherein each reaction chamber comprises one of the reaction mixtures of the plurality of reaction mixtures; and an input chamber fluidically connected between the sample interface and the reaction chambers. In some embodiments, the sample interface, input chamber, and reaction chambers are fluidically connected by one or more valves. In some embodiments, the input chamber comprises lysis reagents.

[0038] Target nucleic acids at different concentrations may be tested with a programmable nuclease-based detection assay as described herein. In some embodiments, the assay is a one-pot (e.g., HotPot) assay. For example, in a one-pot assay, target nucleic acids may be amplified with isothermal amplification methods such as loop mediated amplification (LAMP) and the amplified targets may be detected with gRNA-Cas enzyme complexes. Standard curves may be constructed with time to results (“TTR”) calculated from the DETECTR curves of controls at various concentration as the y-axis and log transformed concentration (e.g., in copies of target nucleic acid) as the x-axis. Assay TTR may be generated for samples of unknown concentrations. The concentrations of the samples may be extrapolated from the standard curve. As a result, the target nucleic acids may be quantified in a one-pot reaction run at a fixed temperature. In some embodiments, the target nucleic acids may be quantified in a one- pot reaction run at variable temperatures (e.g., with a thermocycling amplification). In some embodiments, results may be obtained within 2 hours (e.g., within 90 minutes, 60 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes).

[0039] Various methods for calculating TTR may be used, such as a “consecutive increase method” or a “threshold method”. The consecutive increase method may comprise monitoring fluorescence changes over time and calling the TTR as the time at which a certain number (such as, for example, 4) of consecutively-increasing points have a slope of at least, e.g., 1.05. The threshold method may comprise monitoring fluorescence changes in DETECTR signal over time and calling the TTR as the time at which the DETECTR fluorescence reaches, e.g.,Docket No.203477-779601 30% of its max fluorescence. In some embodiments, the TTR is the time to reach an inflection point in a plot of detected signal over time. Given a sigmoidal shape of a resulting curve, the consecutive increase method may allow for calling an inflection faster than the threshold method, though both methods yield TTRs that may be used for quantitation. Examples of standard curves with different Cas enzymes are disclosed here. In general, whichever method is applied for defining TTR, the same method is applied to each control sample of known concentration to establish a standard curve, and to each test sample compared to the standard curve.

[0040] In some embodiments, a one-pot assay comprises, in a single reaction, reagents for reverse transcribing target RNA (e.g., viral RNA) into DNA, LAMP primers for amplification of a specific target region, a programmable nuclease complexed with a guide nucleic acid (e.g., a guide RNA-Cas enzyme complex) capable of specifically recognizing the amplified target region, and a reporter (e.g., a fluorescent reporter) that is transcollaterally cleaved by the activated programmable nuclease in response to target binding. The increase of the fluorescent signals (e.g., the rate thereof) may be used to determine the TTR. A target nucleic acid that is DNA may be measured in a similar manner, but without need for reverse transcription reagents.

[0041] Generally, a standard curve for a specific target may be generated by measuring TTR of controls at different known concentrations, such as, for example, over 2 logs, 3 logs, 4 logs, 5 logs, 6 logs, 7 logs, or 8 logs. The TTR of controls can be plotted against log transformed copy number. The TTR of a sample of unknown concentration may be determined with the one- pot assay and used to determine the measured amount (e.g., copy number) from the standard curve.

[0042] In some embodiments, a one-pot assay that combines DETECTR with LAMP may be optimized for fast time to results. In some embodiments, in order to achieve resolutions in high sample concentrations to cover 4+ logs of range, the assay speed may be slowed by reducing an amount of the programmable nuclease, using a less catalytically active programmable nuclease, and / or by including additives, such glycerol, sucrose, BSA, or Mg2+, or the like. Examples of standard curves generated using various additives are provided herein.

[0043] In some aspects, described herein is a method for measuring an amount of a target nucleic acid in a sample. In some embodiments, the method comprises: (a) initiating detection reactions by heating the plurality of reaction mixtures to a reaction temperature; (b) measuring the detectable product at each of a plurality of times for each of the plurality of reaction mixtures; (c) determining a time to result (TTR) for each of the plurality of reaction mixtures, wherein the TTR is a reaction time to reach a predetermined state; (d) comparing the TTR for the sample reaction mixture to the TTRs of the control reaction mixtures; and (e) determining anDocket No.203477-779601 amount of the target nucleic acid in the sample reaction mixture based on the comparing.

[0044] In some embodiments, the TTR is a time from initiating the detection reaction to reaching a predetermined rate of increase in formation of the detectable product. In some embodiments, the TTR is a time from initiating the detection reaction to reaching a predetermined percent of a maximum level of detectable product (e.g., formed at a last of the plurality of times). In some embodiments, the predetermined percent is at least 20%, 25%, or 30% of the maximum level.

[0045] In some embodiments, the method further comprises using the TTR measured for each control sample to generate a standard curve for measuring the target nucleic acid in an unknown sample. In some embodiments, the standard curve is about linear on a log scale over a range of amounts of the target nucleic acid per reaction mixture, wherein the range of amounts span at least 2 logs, 3 logs, 4 logs, 5 logs, 6 logs, 7 logs, or 8 logs. In some embodiments, the standard curve is about linear on a log scale over a range of amounts of the target nucleic acid per reaction mixture, wherein the range of amounts comprises 107, 106, 105, 104, 1000, 100, 50, 25, 10, or fewer copies of the target nucleic acid. In some embodiments, the standard curve that is about linear on a log scale has an R2value of at least 0.85, 0.9, or 0.95 over the range of amounts. In some embodiments, the TTRs for an amount within the range of amounts is about 1- 60 minutes, 1-30 minutes, 1-20 minutes, 1-10 minutes, or 1-5 minutes. Non-limiting examples of resulting standard curves from the detection reactions are described herein.

[0046] In some embodiments, the programmable nuclease-based detection reactions comprise amplification reactions to amplify the target nucleic acid. In some embodiments, the amplification reactions comprise loop mediated amplification (LAMP). In some embodiments, the programmable nuclease-based detection reactions comprise reverse transcription reactions. In some embodiments, the programmable nuclease-based detection reactions comprise cleaving the reporter in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid. In some embodiments: (i) the reporter comprises a fluorescent label and a quencher; and (ii) measuring the detectable product comprises measuring a level of fluorescence in the reaction mixtures. Programmable Nucleases

[0047] Disclosed herein are programmable nucleases and uses thereof, e.g., detection and editing of target nucleic acids. In some cases, a programmable nuclease is capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment. A programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence. The programmable nuclease can be activated upon binding of theDocket No.203477-779601 guide nucleic acid to its target nucleic acid and can non-specifically degrade a non-target nucleic acid in its environment. The programmable nuclease has trans cleavage activity once activated. A programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease or Cas effector protein). A guide nucleic acid (e.g., crRNA) and Cas protein can form a CRISPR enzyme (also referred to herein as a programmable nuclease complex or probe).

[0048] In some embodiments, one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule). A programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter. The programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter. Such a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein can be activated by a target RNA or a target DNA. For example, a programmable nuclease, e.g., a Cas enzyme, can be activated by a target RNA nucleic acid or a target DNA nucleic acid to cleave RNA reporters. In some embodiments, the programmable nuclease can bind to a target ssDNA which initiates trans cleavage of RNA reporters. In some instances, a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA- activated programmable DNA nuclease.

[0049] As used herein, a programmable nuclease generally refers to any enzyme that can cleave nucleic acid. The programmable nuclease can be any enzyme that can be or has been designed, modified, or engineered by human contribution so that the enzyme targets or cleaves the nucleic acid in a sequence-specific manner. Programmable nucleases can include, for example, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and / or RNA-guided nucleases such as the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) nucleases or Cpfl. Programmable nucleases can also include, for example, PfAgo and / or NgAgo.

[0050] ZFNs can cut genetic material in a sequence- specific matter and can be designed, or programmed, to target specific viral targets. A ZFN is composed of two domains: a DNA- binding zinc-finger protein linked to the Fokl nuclease domain. The DNA-binding zinc-finger protein is fused with the non-specific Fokl cleave domain to create ZFNs. The protein will typically dimerize for activity. Two ZFN monomers form an active nuclease; each monomer binds to adjacent half- sites on the target. The sequence specificity of ZFNs is determined by ZFPs. Each zinc-finger recognizes a 3-bp DNA sequence, and 3-6 zinc-fingers are used toDocket No.203477-779601 generate a single ZFN subunit that binds to DNA sequences of 9-18 bp. The DNA-binding specificities of zinc-fingers is altered by mutagenesis. New ZFPs are programmed by modular assembly of pre-characterized zinc fingers.

[0051] Transcription activator-like effector nucleases (TALENs) can cut genetic material in a sequence-specific matter and can be designed, or programmed, to target specific viral targets. TALENs contain the Fokl nuclease domain at their carboxyl termini and a class of DNA binding domains known as transcription activator- like effectors (TALEs). TALENs are composed of tandem arrays of 33-35 amino acid repeats, each of which recognizes a single base- pair in the major groove of target viral DNA. The nucleotide specificity of a domain comes from the two amino acids at positions 12 and 13 where Asn-Asn, Asn-Ile, His-Asp and Asn-Gly recognize guanine, adenine, cytosine and thymine, respectively. That pattern allows one to program TALENs to target various nucleic acids.

[0052] The programmable nuclease can comprise any type of engineered enzyme. Alternatively, the programmable nuclease can comprise CRISPR enzymes derived from naturally occurring bacteria or phage. A programmable nuclease can be a Cas effector protein (also referred to, interchangeably, as a Cas nuclease). A guide nucleic acid (e.g., a crRNA) and Cas effector protein can form a CRISPR enzyme. The programmable nuclease can be a CRISPR- Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid. The programmable nuclease can comprise one or more amino acid modifications. The programmable nuclease can be a nuclease derived from a CRISPR-Cas system. The programmable nuclease can be a nuclease derived from recombineering. In some embodiments, the programmable nuclease further comprises a Cas enzyme. In some embodiments, the Cas enzyme is selected from the group consisting of Cas12, Cas13, Cas14, Cas14a, Cas14a1, and CasPhi.

[0053] In some cases, the programmable nuclease is Cas13. Sometimes the Cas13 is Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, or Cas13f. In some cases, the programmable nuclease is Mad7 or Mad2. In some cases, the programmable nuclease is Cas12. Sometimes the Cas12 is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, or Cas12k. In some cases, the Cas12 can be a Cas12 variant having the sequence of SEQ ID NO: 17 (or a sequence with at least 60%, 70%, 80%, 90%, or 95% identity thereto). In some cases, the programmable nuclease is Csm1, Cas9, C2c4, C2c8, C2c5, C2c10, C2c9, or CasZ. Sometimes, the Csm1 is also called smCms1, miCms1, obCms1, or suCms1. Sometimes Cas13a is also called C2c2. Sometimes CasZ is also called Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. Sometimes, the programmable nuclease is a type V CRISPR-Cas system. InDocket No.203477-779601 some cases, the programmable nuclease is a type VI CRISPR-Cas system. Sometimes the programmable nuclease is a type III CRISPR-Cas system. In some embodiments, the programmable nuclease can be an engineered nuclease that is not from a naturally occurring CRISPR-Cas system. In some cases, the programmable nuclease is from at least one of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichia buccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rca), Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr), Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listeria newyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp. (Psm), Capnocytophaga canimorsus (Cca Lachnospiraceae bacterium (Lba), Bergeyella zoohelcum (Bzo), Prevotella intermedia (Pin), Prevotella buccae (Pbu), Alistipes sp. (Asp), Riemerella anatipestifer (Ran), Prevotella aurantiaca (Pau), Prevotella saccharolytica (Psa), Prevotella intermedia (Pin2), Capnocytophaga canimorsus (Cca), Porphyromonas gulae (Pgu), Prevotella sp. (Psp), Porphyromonas gingivalis (Pig), Prevotella intermedia (Pin3), Enterococcus italicus (Ei), Lactobacillus salivarius (Ls), or Thermus thermophilus (Tt). Sometimes the Cas13 is at least one of LbuCas13a, LwaCas13a, LbaCas13a, HheCas13a, PprCas13a, EreCas13a, CamCas13a, or LshCas13a. The trans cleavage activity of the CRISPR enzyme can be activated when the crRNA is complexed with the target nucleic acid. The trans cleavage activity of the CRISPR enzyme can be activated when the guide nucleic acid comprising a tracrRNA and crRNA are complexed with the target nucleic acid. The target nucleic acid can be RNA or DNA.

[0054] Disclosed herein are programmable nucleases and uses thereof, e.g., detection and editing of target nucleic acids. In some instances, programmable nucleases comprise a Type V CRISPR / Cas protein. In some instances, Type V CRISPR / Cas proteins comprise nucleic acid cleavage activity. In some instances, Type V CRISPR / Cas proteins cleave or nick single- stranded nucleic acids, double, stranded nucleic acids, or a combination thereof. In some cases, Type V CRISPR / Cas proteins cleave single-stranded nucleic acids. In some cases, Type V CRISPR / Cas proteins cleave double-stranded nucleic acids. In some cases, Type V CRISPR / Cas proteins nick double-stranded nucleic acids. Typically, guide nucleic acids of Type V CRISPR / Cas proteins hybridize to ssDNA or dsDNA. However, the trans cleavage activity of Type V CRISPR / Cas protein is typically directed towards ssDNA. In some cases, the Type V CRISPR / Cas protein comprises a catalytically inactive nuclease domain. A catalytically inactive domain of a Type V CRISPR / Cas protein may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to a wild type nuclease domain of the Type V CRISPR / Cas protein. Said mutations may be present within a cleaving or active site of the nuclease.

[0055] In some instances, the Type V Cas protein is a CasФ protein. A CasФ protein can function as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleicDocket No.203477-779601 acid. A programmable CasФ nuclease may have a single active site in a RuvC domain that is capable of catalyzing pre-crRNA processing and nicking or cleaving of nucleic acids. This compact catalytic site may render the programmable CasФ nuclease especially advantageous for genome engineering and new functionalities for genome manipulation.

[0056] In some instances, the programmable nuclease is a Type VI Cas protein. In some embodiments, the Type VI Cas protein is a programmable Cas13 nuclease. The general architecture of a Cas13 protein includes an N-terminal domain and two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains separated by two helical domains. The HEPN domains each comprise aR-X4-H motif. Shared features across Cas13 proteins include that upon binding of the crRNA of the guide nucleic acid to a target nucleic acid, the protein undergoes a conformational change to bring together the HEPN domains and form a catalytically active RNase. Thus, two activatable HEPN domains are characteristic of a programmable Cas13 nuclease of the present disclosure. However, programmable Cas13 nucleases also consistent with the present disclosure include Cas13 nucleases comprising mutations in the HEPN domain that enhance the Cas13 proteins cleavage efficiency or mutations that catalytically inactivate the HEPN domains. Programmable Cas13 nucleases consistent with the present disclosure also Cas13 nucleases comprising catalytic components. In some instances, the Cas effector is a Cas 13 effector. In some instances, the Cas13 effector is a Cas13a, a Cas13b, a Cas 13c, a Cas 13d, or a Cas 13e effector protein.

[0057] In some embodiments, the programmable nuclease comprises a Cas12 protein, wherein the Cas12 enzyme binds and cleaves double stranded DNA and single stranded DNA. In some embodiments, programmable nuclease comprises a Cas13 protein, wherein the Cas13 enzyme binds and cleaves single stranded RNA. In some embodiments, programmable nuclease comprises a Cas14 protein, wherein the Cas14 enzyme binds and cleaves both double stranded DNA and single stranded DNA.

[0058] Table 1 provides illustrative amino acid sequences of programmable nucleases having trans-cleavage activity. In some instances, programmable nucleases described herein comprise an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID Nos: 1-72. The programmable nuclease may consist of an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one or SEQ ID Nos: 1- 72. The programmable nuclease may comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of any one of SEQ ID NOs:Docket No.203477-779601 -72. TABLE 1 – Example Protein SequencesDocket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601Docket No.203477-779601

[0059] Other Exemplary protein sequences are described in the following applications: PCT / US2021 / 033271, PCT / US2021 / 035031, PCT / US2022 / 028865, PCT / US2022 / 034110, and PCT / US2022 / 034596, all of which are herein incorporated by reference in their entireties.

[0060] In some instances, the Type V CRISPR / Cas protein has been modified (also referred to as an engineered protein). For example, a Type V CRISPR / Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS). In some cases, the NLS may comprise a sequence of KRPAATKKAGQAKKKKEF. Type V CRISPR / Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the Type V CRISPR / Cas protein is codon optimized for a human cell.

[0061] In some cases, the effector proteins comprise a RuvC domain (e.g., a partial RuvC domain). In some instances, the RuvC domain may be defined by a single, contiguous sequence, or a set of partial RuvC domains that are not contiguous with respect to the primary amino acid sequence of the protein. An effector protein of the present disclosure may include multipleDocket No.203477-779601 partial RuvC domains, which may combine to generate a RuvC domain with substrate binding or catalytic activity. For example, an effector protein may include three partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the effector protein, but form a RuvC domain once the protein is produced and folds. In some cases, effector proteins comprise a recognition domain with a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. In some instances, the effector protein does not comprise a zinc finger domain. In some instances, the effector protein does not comprise an HNH domain.

[0062] Effector proteins disclosed herein may function as an endonuclease that catalyzes cleavage at a specific position (e.g., at a specific nucleotide within a nucleic acid sequence) in a target nucleic acid. The target nucleic acid may be single stranded RNA (ssRNA), double stranded DNA (dsDNA) or single-stranded DNA (ssDNA). In some instances, the target nucleic acid is single-stranded DNA. In some instances, the target nucleic acid is single-stranded RNA. The effector proteins may provide cis cleavage activity, trans cleavage activity, nickase activity, or a combination thereof. Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide nucleic acid (e.g., a dual gRNA or a sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guide nucleic acid. Trans cleavage activity (also referred to as transcollateral cleavage) is cleavage of ssDNA or ssRNA that is near, but not hybridized to the guide nucleic acid. Trans cleavage activity is triggered by the hybridization of guide nucleic acid to the target nucleic acid. Nickase activity is a selective cleavage of one strand of a dsDNA.

[0063] Effector proteins of the present disclosure, dimers thereof, and multimeric complexes thereof may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some instances, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides of a 5’ or 3’ terminus of a PAM sequence. A target nucleic acid may comprise a PAM sequence adjacent to a sequence that is complementary to a guide nucleic acid spacer region.

[0064] In some instances, the Type V CRISPR / Cas protein has been modified (also referred to as an engineered protein). For example, a Type V CRISPR / Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS). Type V CRISPR / Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the Type V CRISPR / Cas protein is codon optimized for a human cell.

[0065] Several programmable nucleases are consistent with the methods and devices of the present disclosure. For example, Cas proteins are programmable nucleases used in theDocket No.203477-779601 methods and systems disclosed herein. Cas proteins can include any of the known Classes and Types of CRISPR / Cas enzymes. Programmable nucleases disclosed herein include Class 1 Cas proteins, such as the Type I, Type IV, or Type III Cas proteins. Programmable nucleases disclosed herein also include the Class 2 Cas proteins, such as the Type II, Type V, and Type VI Cas proteins. Programmable nucleases included in the devices disclosed herein and methods of use thereof include a Type V or Type VI Cas proteins.

[0066] In some instances, the programmable nuclease is a Type V Cas protein. In general, a Type V Cas effector protein comprises a RuvC domain, but lacks an HNH domain. In most instances, the RuvC domain of the Type V Cas effector protein comprises three patrial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains). In some instances, the three RuvC subdomains are located within the C-terminal half of the Type V Cas effector protein. In some instances, none of the RuvC subdomains are located at the N terminus of the protein. In some instances, the RuvC subdomains are contiguous. In some instances, the RuvC subdomains are not contiguous with respect to the primary amino acid sequence of the Type V Cas protein, but form a RuvC domain once the protein is produced and folds. In some instances, there are zero to about 50 amino acids between the first and second RuvC subdomains. In some instances, there are zero to about 50 amino acids between the second and third RuvC subdomains. In some instances, the Cas effector is a Cas14 effector. In some instances, the Cas14 effector is a Cas14a, Cas14a1, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, or Cas14u effector. In some instances, the Cas effector is a CasPhi effector. In some instances, the Cas effector is a Cas12 effector. In some instances, the Cas12 effector is a Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, or Cas12j effector.

[0067] In some instances, the Type V CRISPR / Cas protein comprises a Cas14 protein. Cas14 proteins may comprise a bilobed structure with distinct amino-terminal and carboxy- terminal domains. The amino- and carboxy-terminal domains may be connected by a flexible linker. The flexible linker may affect the relative conformations of the amino- and carboxyl- terminal domains. The flexible linker may be short, for example less than 10 amino acids, less than 8 amino acids, less than 6 amino acids, less than 5 amino acids, or less than 4 amino acids in length. The flexible linker may be sufficiently long to enable different conformations of the amino- and carboxy-terminal domains among two Cas14 proteins of a Cas14 dimer complex (e.g., the relative orientations of the amino- and carboxy-terminal domains differ between two Cas14 proteins of a Cas14 homodimer complex). The linker domain may comprise a mutation which affects the relative conformations of the amino- and carboxyl-terminal domains. The linker may comprise a mutation which affects Cas14 dimerization. For example, a linker mutation may enhance the stability of a Cas14 dimer.Docket No.203477-779601

[0068] In some instances, the amino-terminal domain of a Cas14 protein comprises a wedge domain, a recognition domain, a zinc finger domain, or any combination thereof. The wedge domain may comprise a multi-strand β-barrel structure. A multi-strand β-barrel structure may comprise an oligonucleotide / oligosaccharide-binding fold that is structurally comparable to those of some Cas12 proteins. The recognition domain and the zinc finger domain may each (individually or collectively) be inserted between β-barrel strands of the wedge domain. The recognition domain may comprise a 4-α-helix structure, structurally comparable but shorter than those found in some Cas12 proteins. The recognition domain may comprise a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. In some cases, a REC lobe may comprise a binding affinity for a PAM sequence in the target nucleic acid. The amino-terminal may comprise a wedge domain, a recognition domain, and a zinc finger domain. The carboxy-terminal may comprise a RuvC domain, a zinc finger domain, or any combination thereof. The carboxy-terminal may comprise one RuvC and one zinc finger domain.

[0069] Cas14 proteins may comprise a RuvC domain or a partial RuvC domain. The RuvC domain may be defined by a single, contiguous sequence, or a set of partial RuvC domains that are not contiguous with respect to the primary amino acid sequence of the Cas14 protein. In some instances, a partial RuvC domain does not have any substrate binding activity or catalytic activity on its own. A Cas14 protein of the present disclosure may include multiple partial RuvC domains, which may combine to generate a RuvC domain with substrate binding or catalytic activity. For example, a Cas14 may include 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the Cas14 protein, but form a RuvC domain once the protein is produced and folds. A Cas14 protein may comprise a linker loop connecting a carboxy terminal domain of the Cas14 protein with the amino terminal domain of the Cas 14 protein, and wherein the carboxy terminal domain comprises one or more RuvC domains and the amino terminal domain comprises a recognition domain.

[0070] Cas14 proteins may comprise a zinc finger domain. In some instances, a carboxy terminal domain of a Cas14 protein comprises a zinc finger domain. In some instances, an amino terminal domain of a Cas14 protein comprises a zinc finger domain. In some instances, the amino terminal domain comprises a wedge domain (e.g., a multi-β-barrel wedge structure), a zinc finger domain, or any combination thereof. In some cases, the carboxy terminal domain comprises the RuvC domains and a zinc finger domain, and the amino terminal domain comprises a recognition domain, a wedge domain, and a zinc finger domain.

[0071] Cas14 proteins may be relatively small compared to many other Cas proteins, making them suitable for nucleic acid detection or gene editing. For instance, a Cas14 proteinDocket No.203477-779601 may be less likely to adsorb to a surface or another biological species due to its small size. The smaller nature of these proteins also allows for them to be more easily packaged as a reagent in a system or assay, and delivered with higher efficiency as compared to other larger Cas proteins. In some cases, a Cas14 protein is 400 to 800 amino acid residues long, 400 to 600 amino acid residues long, 440 to 580 amino acid residues long, 460 to 560 amino acid residues long, 460 to 540 amino acid residues long, 460 to 500 amino acid residues long, 400 to 500 amino acid residues long, or 500 to 600 amino acid residues long. In some cases, a Cas14 protein is less than about 550 amino acid residues long. In some cases, a Cas14 protein is less than about 500 amino acid residues long.

[0072] In some instances, a Cas14 protein may function as an endonuclease that catalyzes cleavage at a specific position within a target nucleic acid. In some instances, a Cas14 protein is capable of catalyzing non-sequence-specific cleavage of a single stranded nucleic acid. In some cases, a Cas14 protein is activated to perform trans cleavage activity after binding of a guide nucleic acid with a target nucleic acid. This trans cleavage activity is also referred to as “collateral” or “transcollateral” cleavage. Trans cleavage activity may be non-specific cleavage of nearby single-stranded nucleic acid by the activated programmable nuclease, such as trans cleavage of reporters with a detection moiety.

[0073] In some embodiments, the Type V CRISPR / Cas enzyme is a programmable Cas12 nuclease. Type V CRISPR / Cas enzymes (e.g., Cas12 or Cas14) lack an HNH domain. A Cas12 nuclease of the present disclosure cleaves a nucleic acid via a single catalytic RuvC domain. The RuvC domain is within a nuclease, or “NUC” lobe of the protein, and the Cas12 nucleases further comprise a recognition, or “REC” lobe. The REC and NUC lobes are connected by a bridge helix and the Cas12 proteins additionally include two domains for PAM recognition termed the PAM interacting (PI) domain and the wedge (WED) domain. In some instances, a programmable Cas12 nuclease can be a Cas12a protein, a Cas12b protein, Cas12c protein, Cas12d protein, or a Cas12e protein.

[0074] In some embodiments, a programmable nuclease as disclosed herein is an RNA- activated programmable RNA nuclease. In some embodiments, a programmable nuclease as disclosed herein is a DNA-activated programmable RNA nuclease. In some embodiments, a programmable nuclease is capable of being activated by a target RNA to initiate trans cleavage of an RNA reporter and is capable of being activated by a target DNA to initiate trans cleavage of an RNA reporter, such as a Type VI CRISPR / Cas enzyme (e.g., a Cas13 nuclease). For example, Cas13a of the present disclosure can be activated by a target RNA to initiate trans cleavage activity of the Cas13a for the cleavage of an RNA reporter and can be activated by a target DNA to initiate trans cleavage activity of the Cas13a for trans cleavage of an RNADocket No.203477-779601 reporter. An RNA reporter can be an RNA-based reporter. In some embodiments, the Cas13a recognizes and detects ssDNA to initiate transcleavage of RNA reporters. Multiple Cas13a isolates can recognize, be activated by, and detect target DNA, including ssDNA, upon hybridization of a guide nucleic acid with the target DNA. For example, Lbu-Cas13a and Lwa- Cas13a can both be activated to transcollaterally cleave RNA reporters by target DNA. Thus, Type VI CRISPR / Cas enzyme (e.g., a Cas13 nuclease, such as Cas13a) can be DNA-activated programmable RNA nucleases, and therefore can be used to detect a target DNA using the methods as described herein. DNA-activated programmable RNA nuclease detection of ssDNA can be robust at multiple pH values. For example, target ssDNA detection by Cas13 can exhibit consistent cleavage across a wide range of pH conditions, such as from a pH of 6.8 to a pH of 8.2. In contrast, target RNA detection by Cas13 can exhibit high cleavage activity of pH values from 7.9 to 8.2. In some embodiments, a DNA-activated programmable RNA nuclease that also is capable of being an RNA-activated programmable RNA nuclease, can have DNA targeting preferences that are distinct from its RNA targeting preferences. For example, the optimal ssDNA targets for Cas13a have different properties than optimal RNA targets for Cas13a. As one example, gRNA performance on ssDNA can not necessarily correlate with the performance of the same gRNAs on RNA. As another example, gRNAs can perform at a high level regardless of target nucleotide identity at a 3’ position on a target RNA sequence. In some embodiments, gRNAs can perform at a high level in the absence of a G at a 3’ position on a target ssDNA sequence. Furthermore, target DNA detected by Cas13 disclosed herein can be directly taken from organisms or can be indirectly generated by nucleic acid amplification methods, such as PCR and LAMP or any amplification method described herein. Key steps for the sensitive detection of a target DNA, such as a target ssDNA, by a DNA-activated programmable RNA nuclease, such as Cas13a, can include: (1) production or isolation of DNA to concentrations above about 0.1 nM per reaction for in vitro diagnostics, (2) selection of a target sequence with the appropriate sequence features to enable DNA detection as these features are distinct from those required for RNA detection, and (3) buffer composition that enhances DNA detection. Engineered programmable nuclease probes

[0075] Disclosed herein are non-naturally occurring compositions and systems comprising at least one of an engineered Cas protein and an engineered guide nucleic acid, which may simply be referred to herein as a Cas protein and a guide nucleic acid, respectively. In general, an engineered Cas protein and an engineered guide nucleic acid refer to a Cas protein and a guide nucleic acid, respectively, that are not found in nature. In some instances, systems and compositions comprise at least one non-naturally occurring component. For example,Docket No.203477-779601 compositions and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. In some instances, compositions and systems comprise at least two components that do not naturally occur together. For example, compositions and systems may comprise a guide nucleic acid comprising a repeat region and a spacer region which do not naturally occur together. Also, by way of example, composition and systems may comprise a guide nucleic acid and a Cas protein that do not naturally occur together. Conversely, and for clarity, a Cas protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes Cas proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.

[0076] In some instances, the guide nucleic acid may comprise a non-natural nucleobase sequence. In some instances, the non-natural sequence is a nucleobase sequence that is not found in nature. The non-natural sequence may comprise a portion of a naturally occurring sequence, wherein the portion of the naturally occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence. In some instances, the guide nucleic acid may comprise two naturally occurring sequences arranged in an order or proximity that is not observed in nature. In some instances, compositions and systems comprise a ribonucleotide complex comprising a CRISPR / Cas effector protein and a guide nucleic acid that do not occur together in nature. Engineered guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together. For example, an engineered guide nucleic acid may comprise a sequence of a naturally occurring repeat region and a spacer region that is complementary to a naturally occurring eukaryotic sequence. The engineered guide nucleic acid may comprise a sequence of a repeat region that occurs naturally in an organism and a spacer region that does not occur naturally in that organism. An engineered guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. The guide nucleic acid may comprise a third sequence disposed at a 3’ or 5’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. For example, an engineered guide nucleic acid may comprise a naturally occurring crRNA and tracrRNA coupled by a linker sequence.

[0077] In some instances, compositions and systems described herein comprise an engineered Cas protein that is similar to a naturally occurring Cas protein. The engineered Cas protein may lack a portion of the naturally occurring Cas protein. The Cas protein may comprise a mutation relative to the naturally-occurring Cas protein, wherein the mutation is not found in nature. The Cas protein may also comprise at least one additional amino acid relative to theDocket No.203477-779601 naturally-occurring Cas protein. For example, the Cas protein may comprise an addition of a nuclear localization signal relative to the natural occurring Cas protein. In certain embodiments, the nucleotide sequence encoding the Cas protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.

[0078] An engineered protein may comprise a modified form of a wildtype counterpart protein. The modified form of the wildtype counterpart may comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the programmable nuclease. For example, a nuclease domain (e.g., RuvC domain) of a Type V CRISPR / Cas protein may be deleted or mutated so that it is no longer functional or comprises reduced nuclease activity. The modified form of the programmable nuclease may have less than 90 %, less than 80 %, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type counterpart. Engineered proteins may have no substantial nucleic acid-cleaving activity. Engineered proteins may be enzymatically inactive or “dead,” that is it may bind to a nucleic acid but not cleave it. An enzymatically inactive protein may comprise an enzymatically inactive domain (e.g., inactive nuclease domain). Enzymatically inactive may refer to an activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to the wild-type counterpart. A dead protein may associate with an engineered guide nucleic acid to activate or repress transcription of a target nucleic acid sequence. In some embodiments, the enzymatically inactive protein is fused with a protein comprising recombinase activity.

[0079] In some instances, compositions and systems provided herein comprise a multi- vector system encoding a Cas protein and a guide nucleic acid described herein, wherein the guide nucleic acid and the Cas protein are encoded by the same or different vectors. In some embodiments, the engineered guide and the engineered Cas protein are encoded by different vectors of the system. Programmable nuclease fusion proteins

[0080] Described herein are various embodiments of programmable nuclease fusion proteins. In some embodiments, a programmable nuclease may be fused with a heterologous polypeptide to form a programmable nuclease fusion protein. In some instances, “fusion partner” can refer to a polypeptide that is fused to another polypeptide. In some embodiments, the heterologous polypeptide may be an enzyme, wherein the enzyme is configured to react an enzyme substrate to generate a detectable signal. In some embodiments, the heterologous polypeptide may provide a tag (i.e., the heterologous polypeptide is a detectable label) for ease ofDocket No.203477-779601 tracking and / or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).

[0081] “Heterologous” as used herein, can refer to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. In some cases, a heterologous polypeptide may comprise a sequence different from a sequence of a given protein. For example, a sequence of an HRP enzyme may be considered heterologous to a sequence of a programmable nuclease when the sequence of the HRP enzyme is different from the sequence of the programmable nuclease. In some embodiments, a programmable nuclease (e.g., a Cas12 protein) can be fused to an active domain from a non-programmable nuclease protein (e.g., an HRP), and the sequence of the active domain may be considered to be a heterologous polypeptide (i.e., it is heterologous to the programmable nuclease).

[0082] In some instances, a programmable nuclease is a fusion protein, wherein the fusion protein comprises a protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-72. In some instances, the fusion protein comprises a programmable nuclease and a fusion partner protein.

[0083] A fusion partner protein is also simply referred to herein as a fusion partner. In some cases, the fusion partner promotes the formation of a multimeric complex of the programmable nuclease. In some cases, the fusion partner is an additional programmable nuclease. In some cases, the multimeric complex comprising the programmable nuclease and the additional programmable nuclease binds a guide nucleic acid. The programmable nucleases of the multimeric complex may bind the guide nucleic acid in an asymmetric fashion. In some cases, one programmable nuclease of the multimeric complex interacts more strongly with the guide nucleic acid than the additional programmable nuclease of the multimeric complex. In some cases, a programmable nuclease interacts more strongly with a target nucleic acid when it is complexed with the guide nucleic acid relative to when the programmable nuclease or the multimeric complex is not complexed with the guide nucleic acid.

[0084] In some instances, fusion partners include, but are not limited to, a protein that directly and / or indirectly provides for increased or decreased transcription and / or translation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription and / or translation regulator, a translation-regulating protein, etc.). In some instances, fusion partners that increase or decrease transcription include a transcription activator domain or a transcription repressor domain, respectively.

[0085] In some cases, a terminus of the programmable nuclease is linked to a terminus ofDocket No.203477-779601 the fusion partner through an amide bond. In some cases, a programmable nuclease is coupled to a fusion partner via a linker protein. In some cases, a programmable nuclease is coupled to a fusion partner via a linker protein. The linker protein may have any of a variety of amino acid sequences. A linker protein may comprise a region of rigidity (e.g., beta sheet, alpha helix), a region of flexibility, or any combination thereof. In some instances, the linker comprises small amino acids, such as glycine and alanine, that impart high degrees of flexibility. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element may include linkers that are all or partially flexible, such that the linker may include a flexible linker as well as one or more portions that confer less flexible structure. Suitable linkers include proteins of 4 linked amino acids to 40 linked amino acids in length, or between 4 linked amino acids and 25 linked amino acids in length. These linkers may be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or may be encoded by a nucleic acid sequence encoding a fusion protein (e.g., an programmable nuclease coupled to a fusion partner). Examples of linker proteins include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn, GGSGGSn, and GGGSn, where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers. Exemplary linkers may comprise amino acid sequences including, but not limited to, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, and GSSSG. Thermostable programmable nuclease

[0086] Described herein are various embodiments of thermostable programmable nucleases. In some embodiments, a programmable nuclease is referred to as an effector protein. An effector protein may be thermostable. In some instances, known effector proteins (e.g., Cas12 nucleases) are relatively thermo-sensitive and only exhibit activity (e.g., cis and / or trans cleavage) sufficient to produce a detectable signal in a diagnostic assay at temperatures less than 40° C, and optimally at about 37° C. A thermostable protein may have enzymatic activity, stability, or folding comparable to those at 37 °C. In some instances, the trans cleavage activity (e.g., the maximum trans cleavage rate as measured by fluorescent signal generation) of an effector protein in a trans cleavage assay at 40 °C may be at least 50% of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold,Docket No.203477-779601 at least 50-fold or more of that at 37 °C.

[0087] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0088] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0089] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0090] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-Docket No.203477-779601 fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0091] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0092] In some instances, the trans cleavage activity may be measured against a negative control in a trans cleavage assay. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 37 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 37 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 40 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 40 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 45 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-Docket No.203477-779601 fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 45 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 50 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 50 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 55 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 55 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 60 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 60 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 65 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at leastDocket No.203477-779601 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 65 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid.

[0093] The reporters described herein can be RNA reporters. The RNA reporters can comprise at least one ribonucleic acid and a detectable moiety. In some embodiments, a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect ssDNA and, further, can specifically trans-cleave RNA reporters. The detection of the target nucleic acid in the sample can indicate the presence of the disease (or disease-causing agent) in the sample and can provide information for taking action to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual.

[0094] Cleavage of a reporter (e.g., a protein-nucleic acid) can produce a signal. The signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample. In some cases, cleavage of the protein-nucleic acid can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. Various devices and / or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample. The sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.

[0095] In an aspect, the present disclosure provides a method for target detection. TheDocket No.203477-779601 method can comprise sample collection. The method can further comprise sample preparation. The method can further comprise detection of one or more target nucleic acids in the collected and prepared sample.

[0096] In another aspect, the present disclosure provides a nucleic acid detection device for target detection. The nucleic acid detection device can be configured for multiplexed target detection. The nucleic acid detection device can be used to collect one or more samples, prepare or process the one or more samples for detection, and optionally divide the one or more samples into a plurality of droplets, aliquots, or subsamples for amplification of one or more target sequences or target nucleic acids. The target sequences may comprise, for example, a biological sequence. The biological sequence can comprise a nucleic acid sequence or an amino acid sequence. In some embodiments, the target sequences are associated with an organism of interest, a disease of interest, a disease state of interest, a phenotype of interest, a genotype of interest, or a gene of interest.

[0097] The nucleic acid detection device can be configured to amplify target nucleic acids contained within the plurality of droplets, aliquots, or subsamples. The nucleic acid detection device can be configured to amplify the target sequences or target nucleic acids contained within the plurality of droplets by individually processing each of the plurality of droplets (e.g., by using a thermocycling process or any other suitable amplification process as described in greater detail below). In some cases, the plurality of droplets can undergo separate thermocycling processes. In some cases, the thermocycling processes can occur simultaneously. In other cases, the thermocycling processes can occur at different times for each droplet.

[0098] The nucleic acid detection device can be further configured to remix the droplets, aliquots, or subsamples after the target nucleic acids in each of the droplets undergo amplification. The nucleic acid detection device can be configured to provide the remixed sample comprising the droplets, aliquots, or subsamples to a detection chamber of the device. The detection chamber can be configured to direct the remixed droplets, aliquots, or subsamples to a plurality of programmable nuclease probes. The detection chamber can be configured to direct the remixed droplets, aliquots, or subsamples along one or more fluid flow paths such that the remixed droplets, aliquots, or subsamples are positioned adjacent to and / or in contact with the one or more programmable nuclease probes. In some cases, the detection chamber can be configured to recirculate or recycle the remixed droplets, aliquots, or subsamples such that the remixed droplets, aliquots, or subsamples are repeatedly placed in contact with one or more programmable nuclease probes over a predetermined period of time.

[0099] The nucleic acid detection device can comprise one or more sensors. The one or more sensors of the nucleic acid detection device can be configured to detect one or more signalsDocket No.203477-779601 that are generated after one or more programmable nucleases of the one or more programmable nuclease probes become activated due to a binding of a guide nucleic acid of the programmable nuclease probes with a target nucleic acid present in the sample. As described elsewhere herein, the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity. Trans cleavage activity can be a non-specific cleavage of nearby single- stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of target nucleic acids with a detection moiety. Once the target nucleic acids are cleaved by the activated programmable nucleases, the detection moiety can be released or separated from the reporter, thereby generating one or more detectable signals. The one or more sensors of the nucleic acid detection device can be configured to register and / or process the one or more detectable signals to confirm a presence and / or an absence of a particular target (e.g., a target nucleic acid).

[0100] The one or more programmable nuclease probes of the nucleic acid detection device can be configured for multiplexed detection. In some cases, each programmable nuclease probe can be configured to detect a particular target. In other cases, each programmable nuclease probe can be configured to detect a plurality of targets. In some cases, a first programmable nuclease probe can be configured to detect a first target or a first set of targets, and a second programmable nuclease probe can be configured to detect a second target or a second set of targets. In other cases, a first programmable nuclease probe can be configured to detect a first set of targets, and a second programmable nuclease probe can be configured to detect a second set of targets. The programmable nuclease probes of the present disclosure can be used to detect a plurality of different target sequences or target nucleic acids. In any of the embodiments described herein, the sample provided to the nucleic acid detection device can comprise a plurality of target sequences or target nucleic acids. In any of the embodiments described herein, the sample provided to the nucleic acid detection device can comprise multiple classes of target sequences or target nucleic acids. Each class of target sequences or class of target nucleic acids can comprise a plurality of target sequences or target nucleic acids associated with a particular organism, disease state, phenotype, or genotype present within the sample. In some cases, each programmable nuclease probe can be used to detect a particular class of target sequences or a particular class of target nucleic acids associated with a particular organism, disease state, phenotype, or genotype present within the sample. In some cases, two or more programmable nuclease probes can be used to detect different classes of target sequences or different classes of target nucleic acids. In such cases, the two or more programmable nuclease probes can comprise different sets or classes of guide nucleic acids complexed to the programmable nucleases of the probes.

[0101] In any of the embodiments described herein, the nucleic acid detection device canDocket No.203477-779601 comprise a single integrated system that is configured to perform sample collection, sample processing, droplet generation, droplet processing (e.g., amplification of target nucleic acids in droplets), droplet remixing, and / or circulation of the remixed droplets within a detection chamber so that at least a portion of the remixed droplets is placed in contact with one or more programmable nuclease probes coupled to the detection chamber. The nucleic acid detection devices of the present disclosure can be disposable devices configured to perform one or more rapid single reaction or multi-reaction tests to detect a presence and / or an absence of one or more target sequences or target nucleic acids.

[0102] The systems and methods of the present disclosure can be used to detect one or more target sequences or nucleic acids in one or more samples. The one or more samples can comprise one or more target sequences or nucleic acids for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry and are compatible with the reagents and support mediums as described herein. Generally, a sample can be taken from any place where a nucleic acid can be found. Samples can be taken from an individual / human, a non-human animal, or a crop, or an environmental sample can be obtained to test for presence of a disease, virus, pathogen, cancer, genetic disorder, or any mutation or pathogen of interest. A biological sample can be blood, serum, plasma, lung fluid, exhaled breath condensate, saliva, spit, urine, stool, feces, mucus, lymph fluid, peritoneal , cerebrospinal fluid, amniotic fluid, breast milk, gastric secretions, bodily discharges, secretions from ulcers, pus, nasal secretions, sputum, pharyngeal exudates, urethral secretions / mucus, vaginal secretions / mucus, anal secretion / mucus, semen, tears, an exudate, an effusion, tissue fluid, interstitial fluid (e.g., tumor interstitial fluid), cyst fluid, tissue, or, in some instances, any combination thereof. A sample can be an aspirate of a bodily fluid from an animal (e.g., human, animals, livestock, pet, etc.) or plant. A tissue sample can be from any tissue that can be infected or affected by a pathogen (e.g., a wart, lung tissue, skin tissue, and the like). A tissue sample (e.g., from animals, plants, or humans) can be dissociated or liquified prior to application to detection system of the present disclosure. A sample can be from a plant (e.g., a crop, a hydroponically grown crop or plant, and / or house plant). Plant samples can include extracellular fluid, from tissue (e.g., root, leaves, stem, trunk etc.). A sample can be taken from the environment immediately surrounding a plant, such as hydroponic fluid / water, or soil. A sample from an environment can be from soil, air, or water. In some instances, the environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest. In some instances, the raw sample is applied to the detection system. In some instances, the sample is diluted with a buffer or a fluid or concentrated prior to application to the detection system. In some cases, the sample is contained in no more than about 200 nanolitersDocket No.203477-779601 (nL). In some cases, the sample is contained in about 200 nL. In some cases, the sample is contained in a volume that is greater than about 200 nL and less than about 20 microliters (µL). In some cases, the sample is contained in no more than 20 µl. In some cases, the sample is contained in no more than 1, 5, 10, 15, 20, 25, 30, 3540, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500 µl, or any of value from 1 µl to 500 µl. In some cases, the sample is contained in from 1 µL to 500 µL, from 10 µL to 500 µL, from 50 µL to 500 µL, from 100 µL to 500 µL, from 200 µL to 500 µL, from 300 µL to 500 µL, from 400 µL to 500 µL, from 1 µL to 200 µL, from 10 µL to 200 µL, from 50 µL to 200 µL, from 100 µL to 200 µL, from 1 µL to 100 µL, from 10 µL to 100 µL, from 50 µL to 100 µL, from 1 µL to 50 µL, from 10 µL to 50 µL, from 1 µL to 20 µL, from 10 µL to 20 µL, or from 1 µL to 10 µL. Sometimes, the sample is contained in more than 500 µl.

[0103] In some instances, the sample is taken from a single-cell eukaryotic organism; a plant or a plant cell; an algal cell; a fungal cell; an animal or an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some instances, the sample is taken from nematodes, protozoans, helminths, or malarial parasites. In some cases, the sample may comprise nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell. In some cases, the sample may comprise nucleic acids expressed from a cell.

[0104] The sample used for disease testing can comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein. In some cases, the target sequence is a portion of a nucleic acid. A nucleic acid can be from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA. A nucleic acid can be from 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length. A nucleic acid can be from 10 to 90, from 20 to 80, from 30 to 70, or from 40 to 60 nucleotides in length. A nucleic acid sequence can be from 10 to 95, from 20 to 95, from 30 to 95, from 40 to 95, from 50 to 95, from 60 to 95, from 10 to 75, from 20 to 75, from 30 to 75, from 40 to 75, from 50 to 75, from 5 to 50, from 15 to 50, from 25 to 50, from 35 to 50, or from 45 to 50 nucleotides in length. A nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length. The target nucleic acid can be reverse complementary to a guide nucleic acid. In some cases, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of a guide nucleic acid can be reverse complementary to a target nucleic acid.Docket No.203477-779601

[0105] In some cases, the target sequence is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from an upper respiratory tract infection, a lower respiratory tract infection, or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a hospital acquired infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from sepsis, in the sample. These diseases can include but are not limited to respiratory viruses (e.g., SARS-CoV-2 (i.e., a virus that causes COVID-19), SARS, MERS, influenza, Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Metapneumovirus (hMPV), Human Rhinovirus / Enterovirus, Influenza A, Influenza A / H1, Influenza A / H3, Influenza A / H1-2009, Influenza B, Influenza C, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus) and respiratory bacteria (e.g., Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae). Other viruses include human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic / protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, and Candida albicans. Pathogenic viruses include but are not limited to: respiratory viruses (e.g., adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, MERS), gastrointestinal viruses (e.g., noroviruses, rotaviruses, some adenoviruses, astroviruses), exanthematous viruses (e.g., the virus that causes measles, the virus that causes rubella, the virus that causes chickenpox / shingles, the virus that causes roseola, the virus that causes smallpox, the virus that causes fifth disease, chikungunya virus infection); hepatic viral diseases (e.g., hepatitis A, B, C, D, E); cutaneous viral diseases (e.g., warts (including genital, anal), herpes (including oral, genital, anal), molluscum contagiosum);Docket No.203477-779601 hemmorhagic viral diseases (e.g., Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever); neurologic viruses (e.g., polio, viral meningitis, viral encephalitis, rabies), sexually transmitted viruses (e.g., HIV, HPV, and the like), immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus anthracis, Bortadella pertussis, Burkholderia cepacia, Corynebacterium diphtheriae, Coxiella burnetii, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella longbeachae, Legionella pneumophila, Leptospira interrogans, Moraxella catarrhalis, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria elongate, Neisseria gonorrhoeae, Parechovirus, Pneumococcus, Pneumocystis jirovecii, Cryptococcus neoformans, Histoplasma capsulatum, Haemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. Vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, M. pneumoniae, Enterobacter cloacae, Kiebsiella aerogenes, Proteus vulgaris, Serratia macesens, Enterococcus faecalis, Enterococcus faecium, Streptococcus intermdius, Streptococcus pneumoniae, and Streptococcus pyogenes. Often the target nucleic acid may comprise a sequence from a virus or a bacterium or other agents responsible for a disease that can be found in the sample. In some cases, the target nucleic acid is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at least one of: human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens includeDocket No.203477-779601 viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic / protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include but are not limited to immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Legionella pneumophila, Streptococcus pyogenes, Streptococcus salivarius, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M. pneumoniae. In some cases, the target sequence is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus of bacterium or other agents responsible for a disease in the sample comprising a mutation that confers resistance to a treatment, such as a single nucleotide mutation that confers resistance to antibiotic treatment.

[0106] In some embodiments, the Coronavirus HKU1 sequence is a target of an assay. InDocket No.203477-779601 some embodiments, the Coronavirus NL63 sequence is a target of an assay. In some embodiments, the Coronavirus 229E sequence is a target of an assay. In some embodiments, the Coronavirus OC43 sequence is a target of an assay. In some embodiments, the SARS-CoV-1 sequence is a target of an assay. In some embodiments, the MERS sequence is a target of an assay. In some embodiments, the SARS-CoV-2 sequence is a target of an assay. In some embodiments, the Respiratory Syncytial Virus A sequence is a target of an assay. In some embodiments, the Respiratory Syncytial Virus B sequence is a target of an assay. In some embodiments, the Influenza A sequence is a target of an assay. In some embodiments, the Influenza B sequence is a target of an assay. In some embodiments, the Human Metapneumovirus sequence is a target of an assay. In some embodiments, the Human Rhinovirus sequence is a target of an assay. In some embodiments, the Human Enterovirus sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 1 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 2 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 3 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 4 sequence is a target of an assay. In some embodiments, the Alphacoronavirus genus sequence is a target of an assay. In some embodiments, the Betacoronavirus genus sequence is a target of an assay. In some embodiments, the Sarbecovirus subgenus sequence is a target of an assay. In some embodiments, the SARS- related virus species sequence is a target of an assay. In some embodiments, the Gammacoronavirus Genus sequence is a target of an assay. In some embodiments, the Deltacoronavirus Genus sequence is a target of an assay. In some embodiments, the Influenza B - Victoria V1 sequence is a target of an assay. In some embodiments, the Influenza B - Yamagata Y1 sequence is a target of an assay. In some embodiments, the Influenza A H1 sequence is a target of an assay. In some embodiments, the Influenza A H2 sequence is a target of an assay. In some embodiments, the Influenza A H3 sequence is a target of an assay. In some embodiments, the Influenza A H4 sequence is a target of an assay. In some embodiments, the Influenza A H5 sequence is a target of an assay. In some embodiments, the Influenza A H6 sequence is a target of an assay. In some embodiments, the Influenza A H7 sequence is a target of an assay. In some embodiments, the Influenza A H8 sequence is a target of an assay. In some embodiments, the Influenza A H9 sequence is a target of an assay. In some embodiments, the Influenza A H10 sequence is a target of an assay. In some embodiments, the Influenza A H11 sequence is a target of an assay. In some embodiments, the Influenza A H12 sequence is a target of an assay. In some embodiments, the Influenza A H13 sequence is a target of an assay. In some embodiments, the Influenza A H14 sequence is a target of an assay. In some embodiments, the Influenza A H15 sequence is a target of an assay. In some embodiments, the Influenza A H16Docket No.203477-779601 sequence is a target of an assay. In some embodiments, the Influenza A N1 sequence is a target of an assay. In some embodiments, the Influenza A N2 sequence is a target of an assay. In some embodiments, the Influenza A N3 sequence is a target of an assay. In some embodiments, the Influenza A N4 sequence is a target of an assay. In some embodiments, the Influenza A N5 sequence is a target of an assay. In some embodiments, the Influenza A N6 sequence is a target of an assay. In some embodiments, the Influenza A N7 sequence is a target of an assay. In some embodiments, the Influenza A N8 sequence is a target of an assay. In some embodiments, the Influenza A N9 sequence is a target of an assay. In some embodiments, the Influenza A N10 sequence is a target of an assay. In some embodiments, the Influenza A N11 sequence is a target of an assay. In some embodiments, the Influenza A / H1-2009 sequence is a target of an assay. In some embodiments, the Human endogenous control 18S rRNA sequence is a target of an assay. In some embodiments, the Human endogenous control GAPDH sequence is a target of an assay. In some embodiments, the Human endogenous control HPRT1 sequence is a target of an assay. In some embodiments, the Human endogenous control GUSB sequence is a target of an assay. In some embodiments, the Human endogenous control RNASe P sequence is a target of an assay. In some embodiments, the Influenza A oseltamivir resistance sequence is a target of an assay. In some embodiments, the Human Bocavirus sequence is a target of an assay. In some embodiments, the SARS-CoV-285Δ sequence is a target of an assay. In some embodiments, the SARS-CoV-2 T1001I sequence is a target of an assay. In some embodiments, the SARS- CoV-23675-3677Δ sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P4715L sequence is a target of an assay. In some embodiments, the SARS-CoV-2 S5360L sequence is a target of an assay. In some embodiments, the SARS-CoV-269-70Δ sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Tyr144fs sequence is a target of an assay. In some embodiments, the SARS-CoV-2242-244Δ sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Y453F sequence is a target of an assay. In some embodiments, the SARS-CoV-2 S477N sequence is a target of an assay. In some embodiments, the SARS-CoV-2 E848K sequence is a target of an assay. In some embodiments, the SARS- CoV-2 N501Y sequence is a target of an assay. In some embodiments, the SARS-CoV-2 D614G sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P681R sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P681H sequence is a target of an assay. In some embodiments, the SARS-CoV-2 L21F sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Q27Stop sequence is a target of an assay. In some embodiments, the SARS-CoV-2 M1fs sequence is a target of an assay. In some embodiments, the SARS-CoV-2 R203fs sequence is a target of an assay. In some embodiments, the Human adenovirus - pan assay sequence is a target of an assay. In some embodiments, the BordetellaDocket No.203477-779601 parapertussis sequence is a target of an assay. In some embodiments, the Bordetella pertussis sequence is a target of an assay. In some embodiments, the Chlamydophila pneumoniae sequence is a target of an assay. In some embodiments, the Mycoplasma pneumoniae sequence is a target of an assay. In some embodiments, the Legionella pneumophila sequence is a target of an assay. In some embodiments, the Bordetella bronchoseptica sequence is a target of an assay. In some embodiments, the Bordetella holmesii sequence is a target of an assay. In some embodiments, the Human adenovirus Type A sequence is a target of an assay. In some embodiments, the Human adenovirus Type B sequence is a target of an assay. In some embodiments, the Human adenovirus Type C sequence is a target of an assay. In some embodiments, the Human adenovirus Type D sequence is a target of an assay. In some embodiments, the Human adenovirus Type E sequence is a target of an assay. In some embodiments, the Human adenovirus Type F sequence is a target of an assay. In some embodiments, the Human adenovirus Type G sequence is a target of an assay. In some embodiments, the MERS-CoV sequence is a target of an assay. In some embodiments, the human metapneumovirus sequence is a target of an assay. In some embodiments, the human parainfluenza 1 sequence is a target of an assay. In some embodiments, the human parainfluenza 2 sequence is a target of an assay. In some embodiments, the human parainfluenza 4 sequence is a target of an assay. In some embodiments, the hCoV-OC43 sequence is a target of an assay. In some embodiments, the human parainfluenza 3 sequence is a target of an assay. In some embodiments, the RSV-A sequence is a target of an assay. In some embodiments, the RSV-B sequence is a target of an assay. In some embodiments, the hCoV-229E sequence is a target of an assay. In some embodiments, the hCoV-HKU1 sequence is a target of an assay. In some embodiments, the hCoV-NL63 sequence is a target of an assay. In some embodiments, the Gammacoronavirus sequence is a target of an assay. In some embodiments, the Deltacoronavirus sequence is a target of an assay. In some embodiments, the Alphacoronavirus sequence is a target of an assay. In some embodiments, the Rhinovirus C sequence is a target of an assay. In some embodiments, the Betacoronavirus sequence is a target of an assay. In some embodiments, the Influenza A sequence is a target of an assay. In some embodiments, the Influenza B sequence is a target of an assay. In some embodiments, the SARS-CoV-2 sequence is a target of an assay. In some embodiments, the SARS-CoV-1 sequence is a target of an assay. In some embodiments, the Sarbecovirus subgenus sequence is a target of an assay. In some embodiments, the SARS- related viruses sequence is a target of an assay. In some embodiments, the MS2 sequence is a target of an assay.

[0107] In some embodiments, the assay is directed to one or more target sequences. In some embodiments, a target sequence is a portion of an antimicrobial resistance (AMR) gene,Docket No.203477-779601 such as CTX-M-1, CTX-M-2, CTX-M-25, CTX-M-8, CTX-M-9, or IMP. In some embodiments, a target sequence is a Mycobacterium tuberculosis sequence, such as a portion of IS1081 or IS6110. In some embodiments, a target sequence is an orthopox virus sequence. In some embodiments, a target sequence is a pseudorabies virus sequence. In some embodiments, a target sequence is a Staphylococcus aureus sequence, such as a portion of gyrA or gyrB, or a portion of a S. aureus thermonuclease. In some embodiments, a target sequence is a Stenotrophomonas maltophilia sequence, such as a sequence of S. maltophilia alpha, S. maltophilia beta, or S. maltophilia gamma. In some embodiments, a target sequence is a Bordetalla sp. sequence, such as a sequence of Bordetella bronchoseptica, Bordetella holmesii, Bordetella parapertussis, or Bordetella pertussis. In some embodiments, a target sequence is a Chlamydophila pneumoniae sequence. In some embodiments, a target sequence is a Human adenovirus sequence, such as a sequence of human adenovirus Type A, Type B, Type C, Type D, Type E, Type F, or Type G. In some embodiments, a target sequence is a human bocavirus sequence. In some embodiments, a target sequence is a Legionella pneumophila sequence. In some embodiments, a target sequence is a Mycoplasma pneumoniae sequence. In some embodiments, a target sequence is an Acinetobacter spp. (e.g., A. pitii, A. baumannii, or A. nosocomialis) sequence, such as a portion of gyrB or a 16S-23S ribosomal RNA intergenic spacer sequence. In some embodiments, a target sequence is a Proteus spp. (e.g., P. mirabilis, P. vulgaris, P. penneri, or P. hauseri) sequence, such as a portion of rpoD or 16S. In some embodiments, a target sequence is an Enterobacter spp. (e.g., E. nimipressuralis, E. cloacae, E. asburiae, E. hormaechei, E. kobei, E. ludwigii, or E. mori) sequence, such as a portion of dnaJ, purG, or 16S. In some embodiments, a target sequence is a Bacillus anthracis sequence, such as a portion of pagA or capB. In some embodiments, a target sequence is a Brucella spp. sequence, such as a portion of 23S, bcsp31, or omp2a. In some embodiments, a target sequence is a Coxiella burnetiid sequence, such as a portion of com1 or IS110. In some embodiments, a target sequence is a Francisella tularensis sequence, such as a portion of 16S. In some embodiments, a target sequence is a Rickettsia spp. sequence, such as a portion of 16S, 23S, or 782-17K genus common antigen. In some embodiments, a target sequence is a Yersinia pestis sequence, such as a portion of pMT1, pCD1, or pPCP1. In some embodiments, a target sequence is a A. calcoaceticus sequence, such as a portion of gyrB. In some embodiments, a target sequence is a Francisella tularensis sequence, such as a portion of tul4 or fopA. In some embodiments, a target sequence is a Nocardia spp. sequence, such as a portion of 16S, hsp65, gyrB, secA1, or sodA. In some embodiments, a target sequence is a Cryptococcus spp. sequence, such as a portion of 18S, URA5, ITS, 28S, or CTX1. In some embodiments, a target sequence is an Actinomyces spp. sequence, such as a portion of 16S. In some embodiments, a target sequence is a Streptococcus spp. sequence, such as a portion of 16S,Docket No.203477-779601 tuf, sodA, or rpoB. In some embodiments, a target sequence is an rRNA sequence, such as a portion of 28S rRNA or 18S rRNA. In some embodiments, a target sequence is a coronavirus sequence, such as a sequence of an alphacoronavirus, betacoronavirus, deltacoronavirus, or gammacoronavirus. In some embodiments, a target sequence is a human coronavirus (hCoV) sequence, such as a sequence of hCoV-229E, hCoV-HKU1, hCoV-NL63, hCoV-OC43. In some embodiments, a target sequence is a MERS-CoV sequence. In some embodiments, the sequence is a mammarenavirus sequence, such as a sequence of a Argentinian mammarenavirus (Junin arenavirus), Lassa mammarenavirus, Lujo mammarenavirus (e.g., an L segment or S segment thereof), or Machupo mammarenavirus. In some embodiments, a target sequence is a human metapneumovirus sequence. In some embodiments, a target sequence is a human parainfluenza sequence, such as a sequence of human parainfluenza 1, human parainfluenza 2, human parainfluenza 3, or human parainfluenza 4. In some embodiments, a target sequence is an influenza A virus sequence, such as a sequence of influenza A H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, N1, N2, N3, N4, N5, N6, N7, N8, or N9. In some embodiments, a target sequence is an influenza B sequence, such as a sequence of influenza B- Victoria V1 or influenza B-Yamagata Y1. In some embodiments, a target sequence is a bacteriophage MS2 sequence. In some embodiments, a target sequence is a rhinovirus C sequence. In some embodiments, a target sequence is a respiratory syncytial virus (RSV) sequence, such as a sequence of RSV-A or RSV-B. In some embodiments, a target sequence is a Sarbecovirus sequence. In some embodiments, a target sequence is a severe acute respiratory syndrome coronavirus (SARS-CoV) sequence, such as a sequence of SARS-CoV-1 or SARS- CoV-2. In some embodiments, a target sequence is a portion of a SARS-COV-2 S gene, such as a sequence comprising 144 / 145 wild-type (WT), deletion (del) 144 / 145 (alpha variant), 156 / 157 WT, del156 / 157 (delta variant), 241 / 243 WT, del241 / 243 (beta variant), 69 / 70 WT, del69 / 70 (alpha variant), A570 WT, A570D (alpha variant), A701 WT, A701V (beta variant), D1118 WT, D1118H (alpha variant), D215 WT, D215G (beta variant), D614 WT, D614G (beta variant), D80 WT, D80A (beta variant), E484 WT, E484K (gamma variant), P681 WT, P681H (alpha variant), P681R (delta variant), S982 WT, S982A (alpha variant), T19 WT, T19R (delta variant), T716 WT, T716F (gamma variant). In some embodiments, a target sequence is a SARS-related virus sequence. In some embodiments, a target sequence is a portion of a gene selected from 16S, 18S, 23S, 28S, ACTB, ATP5ME, ATP5MF, ATP5MG, ATP5PB, BCSP31, CAPB, CHMP2A, C1orf43, COM1, CTX1, DNAJ, EMC7, FOPA, GPI, GAPDH, GUSB, GYRB, HRPT1, HSP65, NDUFB3, NDUFB4, NDUFB8, OMP2A, PAGA, PRDX1, PSMB2, PSMB4, PURG, RAB7A, REEP5, RNaseP, RPL13, RPL19, RPL27A, RPL30, RPL31, RPL32, RPL37A, RPOB, RPOD, RPS10, RPS27, RPS29, RPS6, SECA1, SNRPD3, SODA, TUF, TUL4, URA5, VCP, VPS29,Docket No.203477-779601 and YWHAG.

[0108] In some embodiments, the one or more targets may be at a concentration of 1 copy / reaction, at least about 2 copies / reaction, at least about 3 copies / reaction, at least about 4 copies / reaction, at least about 5 copies / reaction, at least about 6 copies / reaction, at least about 7 copies / reaction, at least about 8 copies / reaction, at least about 9 copies / reaction, at least about 10 copies / reaction, at least about 20 copies / reaction, at least about 30 copies / reaction, at least about 40 copies / reaction, at least about 50 copies / reaction, at least about 60 copies / reaction, at least about 70 copies / reaction, at least about 80 copies / reaction, at least about 90 copies / reaction, at least about 100 copies / reaction, at least about 200 copies / reaction, at least about 300 copies / reaction, at least about 400 copies / reaction, at least about 500 copies / reaction, at least about 600 copies / reaction, at least about 700 copies / reaction, at least about 800 copies / reaction, at least about 900 copies / reaction, at least about 1000 copies / reaction, at least about 2000 copies / reaction, at least about 3000 copies / reaction, at least about 4000 copies / reaction, at least about 5000 copies / reaction, at least about 6000 copies / reaction, at least about 7000 copies / reaction, at least about 8000 copies / reaction, at least about 9000 copies / reaction, at least about 10000 copies / reaction, at least about 20000 copies / reaction, at least about 30000 copies / reaction, at least about 40000 copies / reaction, at least about 50000 copies / reaction, at least about 60000 copies / reaction, at least about 70000 copies / reaction, at least about 80000 copies / reaction, at least about 90000 copies / reaction, or at least about 100000 copies / reaction.

[0109] The sample used for cancer testing or cancer risk testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene with a mutation associated with cancer, from a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle. Sometimes, the target nucleic acid encodes for a cancer biomarker, such as a prostate cancer biomarker or non-small cell lung cancer. In some cases, the assay can be used to detect “hotspots” in target nucleic acids that can be predictive of cancer, such as lung cancer, cervical cancer, in some cases, the cancer can be a cancer that is caused by a virus. Some non-limiting examples of viruses that cause cancers in humans include Epstein-Barr virus (e.g., Burkitt’s lymphoma, Hodgkin’s Disease, and nasopharyngeal carcinoma); papillomavirus (e.g., cervical carcinoma, anal carcinoma, oropharyngeal carcinoma, penile carcinoma); hepatitis B and C viruses (e.g., hepatocellular carcinoma); human adult T-cell leukemia virus type 1 (HTLV-1) (e.g., T-cell leukemia); and Merkel cell polyomavirus (e.g., Merkel cell carcinoma). One skilled in the art will recognize that viruses can cause or contributeDocket No.203477-779601 to other types of cancers. In some cases, the target nucleic acid is a portion of a nucleic acid that is associated with a blood fever. In some cases, the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: ALK, APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CASR, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CTNNA1, DICER1, DIS3L2, EGFR, EPCAM, FH, FLCN, GATA2, GPC3, GREM1, HOXB13, HRAS, KIT, MAX, MEN1, MET, MITF, MLH1, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, NTHL1, PALB2, PDGFRA, PHOX2B, PMS2, POLD1, POLE, POT1, PRKAR1A, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RB1, RECQL4, RET, RUNX1, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMAD4, SMARCA4, SMARCB1, SMARCE1, STK11, SUFU, TERC, TERT, TMEM127, TP53, TSC1, TSC2, VHL, WRN, and WT1.

[0110] The sample used for genetic disorder testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. In some embodiments, the genetic disorder is hemophilia, sickle cell anemia, β- thalassemia, Duchene muscular dystrophy, severe combined immunodeficiency, or cystic fibrosis. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder. In some cases, the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: CFTR, FMR1, SMN1, ABCB11, ABCC8, ABCD1, ACAD9, ACADM, ACADVL, ACAT1, ACOX1, ACSF3, ADA, ADAMTS2, ADGRG1, AGA, AGL, AGPS, AGXT, AIRE, ALDH3A2, ALDOB, ALG6, ALMS1, ALPL, AMT, AQP2, ARG1, ARSA, ARSB, ASL, ASNS, ASPA, ASS1, ATM, ATP6V1B1, ATP7A, ATP7B, ATRX, BBS1, BBS10, BBS12, BBS2, BCKDHA, BCKDHB, BCS1L, BLM, BSND, CAPN3, CBS, CDH23, CEP290, CERKL, CHM, CHRNE, CIITA, CLN3, CLN5, CLN6, CLN8, CLRN1, CNGB3, COL27A1, COL4A3, COL4A4, COL4A5, COL7A1, CPS1, CPT1A, CPT2, CRB1, CTNS, CTSK, CYBA, CYBB, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP27A1, DBT, DCLRE1C, DHCR7, DHDDS, DLD, DMD, DNAH5, DNAI1, DNAI2, DYSF, EDA, EIF2B5, EMD, ERCC6, ERCC8, ESCO2, ETFA, ETFDH, ETHE1, EVC, EVC2, EYS, F9, FAH, FAM161A, FANCA, FANCC, FANCG, FH, FKRP, FKTN, G6PC, GAA, GALC, GALK1, GALT, GAMT, GBA, GBE1, GCDH, GFM1, GJB1, GJB2, GLA, GLB1, GLDC, GLE1, GNE, GNPTAB, GNPTG, GNS, GRHPR, HADHA, HAX1, HBA1,, HBA2, HBB, HEXA, HEXB, HGSNAT, HLCS, HMGCL, HOGA1, HPS1, HPS3, HSD17B4, HSD3B2, HYAL1, HYLS1, IDS, IDUA, IKBKAP,Docket No.203477-779601 IL2RG, IVD, KCNJ11, LAMA2, LAMA3, LAMB3, LAMC2, LCA5, LDLR, LDLRAP1, LHX3, LIFR, LIPA, LOXHD1, LPL, LRPPRC, MAN2B1, MCOLN1, MED17, MESP2, MFSD8, MKS1, MLC1, MMAA, MMAB, MMACHC, MMADHC, MPI, MPL, MPV17, MTHFR, MTM1, MTRR, MTTP, MUT, MYO7A, NAGLU, NAGS, NBN, NDRG1, NDUFAF5, NDUFS6, NEB, NPC1, NPC2, NPHS1, NPHS2, NR2E3, NTRK1, OAT, OPA3, OTC, PAH, PC, PCCA, PCCB, PCDH15, PDHA1, PDHB, PEX1, PEX10, PEX12, PEX2, PEX6, PEX7, PFKM, PHGDH, PKHD1, PMM2, POMGNT1, PPT1, PROP1, PRPS1, PSAP, PTS, PUS1, PYGM, RAB23, RAG2, RAPSN, RARS2, RDH12, RMRP, RPE65, RPGRIP1L, RS1, RTEL1, SACS, SAMHD1, SEPSECS, SGCA, SGCB, SGCG, SGSH, SLC12A3, SLC12A6, SLC17A5, SLC22A5, SLC25A13, SLC25A15, SLC26A2, SLC26A4, SLC35A3, SLC37A4, SLC39A4, SLC4A11, SLC6A8, SLC7A7, SMARCAL1, SMPD1, STAR, SUMF1, TAT, TCIRG1, TECPR2, TFR2, TGM1, TH, TMEM216, TPP1, TRMU, TSFM, TTPA, TYMP, USH1C, USH2A, VPS13A, VPS13B, VPS45, VRK1, VSX2, WNT10A, XPA, XPC, and ZFYVE26.

[0111] The sample used for phenotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a phenotypic trait.

[0112] The sample used for genotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a genotype.

[0113] The sample used for ancestral testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a geographic region of origin or ethnic group.

[0114] The sample can be used for identifying a disease status. For example, a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject. The disease can be a cancer or genetic disorder. Sometimes, a method may comprise obtaining a serum sample from a subject; and identifying a disease status of the subject. Often, the disease status is prostate disease status. In any of the embodiments described herein, the device can be configured for asymptomatic, pre-symptomatic, and / or symptomatic diagnostic applications, irrespective of immunity. In any of the embodiments described herein, the device can be configured to perform one or more serological assays on a sample (e.g., a sample comprising blood).Docket No.203477-779601

[0115] In some embodiments, the sample can be used to identify a mutation in a target nucleic acid of a plant or of a bacteria, virus, or microbe associated with a plant or soil. The devices and methods of the present disclosure can be used to identify a mutation of a target nucleic acid that affects the expression of a gene. A mutation that affects the expression of gene can be a mutation of a target nucleic acid within the gene, a mutation of a target nucleic acid comprising RNA associated with the expression of a gene, or a target nucleic acid comprising a mutation of a nucleic acid associated with regulation of expression of a gene, such as an RNA or a promoter, enhancer, or repressor of the gene. Often, the mutation is a single nucleotide mutation

[0116] In some instances, the target nucleic acid is a single stranded nucleic acid. Alternatively, or in combination, the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents. The target nucleic acid can be a RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. The target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA). In some cases, the target nucleic acid is mRNA. In some cases, the target nucleic acid is from a virus, a parasite, or a bacterium described herein. In some cases, the target nucleic acid is transcribed from a gene as described herein.

[0117] A number of target nucleic acids are consistent with the systems and methods disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some cases, the sample has at least 2 target nucleic acids. In some cases, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acids. In some cases, the sample has from 1 to 10,000, from 100 to 8000, from 400 to 6000, from 500 to 5000, from 1000 to 4000, or from 2000 to 3000 target nucleic acids. In some cases, the sample has from 100 to 9500, from 100 to 9000, from 100 to 8500, from 100 to 8000, from 100 to 7500, from 100 to 7000, from 100 to 6500, from 100 to 6000, from 100 to 5500, from 100 to 5000, from 250 to 9500, from 250 to 9000, from 250 to 8500, from 250 to 8000, from 250 to 7500, from 250 to 7000, from 250 to 6500, from 250 to 6000, from 250 to 5500, from 250 to 5000, from 2500 to 9500, from 2500 to 9000, from 2500 to 8500, from 2500 to 8000, from 2500 to 7500, from 2500 to 7000, from 2500 to 6500, from 2500 to 6000, from 2500 to 5500, or from 2500 to 5000 target nucleic acids. In some cases, the method detects target nucleic acid present at least at one copy per 101non-target nucleic acids, 102non-target nucleic acids, 103non-target nucleic acids, 104non-target nucleic acids, 105non- target nucleic acids, 106non-target nucleic acids, 107non-target nucleic acids, 108non-targetDocket No.203477-779601 nucleic acids, 109non-target nucleic acids, or 1010non-target nucleic acids.

[0118] A number of target nucleic acid populations are consistent with the systems and methods disclosed herein. Some methods described herein can be implemented to detect two or more target nucleic acid populations present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 target nucleic acid populations. In some cases, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some cases, the sample has from 3 to 50, from 5 to 40, or from 10 to 25 target nucleic acid populations. In some cases, the sample has from 2 to 50, from 5 to 50, from 10 to 50, from 2 to 25, from 3 to 25, from 4 to 25, from 5 to 25, from 10 to 25, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 10 to 20, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, or from 9 to 10 target nucleic acid populations. In some cases, the methods of the present disclosure can be implemented to detect target nucleic acid populations that are present at least at one copy per 101non-target nucleic acids, 102non-target nucleic acids, 103non-target nucleic acids, 104non-target nucleic acids, 105non-target nucleic acids, 106non- target nucleic acids, 107non-target nucleic acids, 108non-target nucleic acids, 109non-target nucleic acids, or 1010non-target nucleic acids. The target nucleic acid populations can be present at different concentrations or amounts in the sample.

[0119] FIG.6 illustrates an exemplary method for programmable nuclease-based detection. The method can comprise collecting a sample. The sample can comprise any type of sample as described herein. The method can comprise preparing the sample. Sample preparation can comprise one or more sample preparation steps. The one or more sample preparation steps can be performed in any suitable order. The one or more sample preparation steps can comprise physical filtration of non-target materials using a macro filter. The one or more sample preparation steps can comprise nucleic acid purification. The one or more sample preparation steps can comprise lysis. The one or more sample preparation steps can comprise heat inactivation. The one or more sample preparation steps can comprise enzyme (e.g., PK) inactivation. The one or more sample preparation steps can comprise adding one or more enzymes or reagents to prepare the sample for target detection.

[0120] In some embodiments, the sample can be provided manually to a nucleic acid detection device of the present disclosure. For example, a swab sample can be dipped into a solution and the sample / solution can be pipetted into the device. In other embodiments, the sample can be provided via an automated syringe. The automated syringe can be configured to control a flow rate at which the sample is provided to the nucleic acid detection device. The automated syringe can be configured to control a volume of the sample that is provided to the nucleic acid detection device over a predetermined period.Docket No.203477-779601

[0121] In some embodiments, the sample can be provided directly to a nucleic acid detection device of the present disclosure. For example, a swab sample can be inserted into a sample chamber on the nucleic acid detection device.

[0122] The sample can be prepared before one or more targets are detected within the sample. The sample preparation steps described herein can process a crude sample to generate a pure or purer sample. Sample preparation can one or more physical or chemical processes, including, for example, nucleic acid purification, lysis, binding, washing, and / or eluting. In certain instances, sample preparation can comprise the following steps, in any order, including sample collection, nucleic acid purification, heat inactivation, enzyme inactivation, and / or base / acid lysis.

[0123] In some embodiments, nucleic acid purification can be performed on the sample. Purification can comprise disrupting a biological matrix of a cell to release nucleic acids, denaturing structural proteins associated with the nucleic acids (nucleoproteins), inactivating nucleases that can degrade the isolated product (RNase and / or DNase), and / or removing contaminants (e.g., proteins, carbohydrates, lipids, biological or environmental elements, unwanted nucleic acids, and / or other cellular debris).

[0124] In some embodiments, lysis of a collected sample can be performed. Lysis can be performed using a protease (e.g., a Proteinase K or PK enzyme). Exemplary proteases include serine proteases (e.g., Proteinase K, Savinase®, trypsin, Protamex®, etc.), metalloproteinases (e.g., MMP-3, etc.), cysteine proteases (e.g., cathepsin B, papin, etc.), threonine proteases, aspartic proteases (e.g., renin, pepsin, cathepsin D, etc.), glutamic proteases, asparagine peptide lyases, or the like. In some cases, a solution of reagents can be used to lyse the cells in the sample and release the nucleic acids so that they are accessible to the programmable nuclease. Active ingredients of the solution can be chaotropic agents, detergents, salts, and can be of high osmolality, ionic strength, and pH. Chaotropic agents or chaotropes are substances that disrupt the three-dimensional structure in macromolecules such as proteins, DNA, or RNA. One example protocol may comprise a 4 M guanidinium isothiocyanate, 25 mM sodium citrate.2H20, 0.5% (w / v) sodium lauryl sarcosinate, and 0.1 M β-mercaptoethanol), but numerous commercial buffers for different cellular targets can also be used. Alkaline buffers can also be used for cells with hard shells, particularly for environmental samples. Detergents such as sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB) can also be implemented to chemical lysis buffers. Cell lysis can also be performed by physical, mechanical, thermal or enzymatic means, in addition to chemically-induced cell lysis mentioned previously. In some cases, depending on the type of sample, nanoscale barbs, nanowires, acoustic generators, integrated lasers, integrated heaters, and / or microcapillary probes can be used to perform lysis.Docket No.203477-779601

[0125] In certain instances, heat inactivation can be performed on the sample. In some embodiments, a processed / lysed sample can undergo heat inactivation to inactivate, in the lysed sample, the proteins used during lysing (e.g., a PK enzyme or a lysing reagent) and / or other residual proteins in the sample (e.g., RNases, DNases, viral proteins, etc.). In some cases, a heating element integrated into a nucleic acid detection device can be used for heat-inactivation. The heating element can be powered by a battery or another source of thermal or electric energy that is integrated with the nucleic acid detection device.

[0126] In certain instances, enzyme inactivation can be performed on the sample. In some embodiments, a processed / lysed sample can undergo enzyme inactivation to inhibit or inactivate, in the lysed sample, the proteins used during lysing (e.g., a PK enzyme or a lysing reagent) and / or other residual proteins in the sample (e.g., RNases, DNases, etc.). In some cases, a solution of reagents can be used to inactivate one or more enzymes present in the sample. Enzyme inactivation can occur before, during, or after lysis, when lysis is performed. For example, an RNase inhibitor may be included as a lysis reagent to inhibit native RNases within the sample (which might otherwise impair target and / or reporter detection downstream). Exemplary RNase inhibitors include RNAse Inhibitor, Murine (NEB), RnaseIn Plus (Promega), Protector Rnase Inhibitor (Roche), SuperaseIn (Ambion), RiboLock (Thermo), Ribosafe (Bioline), or the like. Alternatively, or in combination, when a protease is used for sample lysis, a protease inhibitor can be applied to the lysed sample to inactivate the protease prior to contacting the sample nucleic acids to the programmable nuclease. Additional application of heat may not be required to inhibit the protease (e.g., proteinase K) sufficiently to prevent additional activity of the protease (which could potentially impair programmable nuclease activity downstream, in some embodiments). Exemplary protease inhibitors include AEBSF, antipain, aprotinin, bestatin, chymostatin, EDTA, leupeptin, pepstatin A, phosphoramidon, PMSF, soybean trypsin inhibitor, TPCK, or the like. In some instances, enzyme inactivation may occur before, during, after, or instead of heat inactivation.

[0127] In some cases, a target nucleic acid within the sample can undergo amplification before binding to a guide nucleic acid, for example a crRNA of a CRISPR enzyme. The target nucleic acid within a purified sample can be amplified. In some instances, amplification can be accomplished using loop mediated amplification (LAMP), isothermal recombinase polymerase amplification (RPA), and / or polymerase chain reaction (PCR). The reagents for nucleic acid amplification can comprise a recombinase, an oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase. The nucleic acid amplification can be transcription mediated amplification (TMA). Nucleic acid amplification can be helicase dependent amplification (HDA) or circular helicase dependent amplification (cHDA). In additional cases,Docket No.203477-779601 nucleic acid amplification is strand displacement amplification (SDA). The nucleic acid amplification can be recombinase polymerase amplification (RPA). The nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR). Nucleic acid amplification is, in some cases, by rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA). The nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes, the nucleic acid amplification is performed for from 1 to 60, from 5 to 55, from 10 to 50, from 15 to 45, from 20 to 40, or from 25 to 35 minutes. Sometimes, the nucleic acid amplification is performed for from 5 to 60, from 10 to 60, from 15 to 60, from 30 to 60, from 45 to 60, from 1 to 45, from 5 to 45, from 10 to 45, from 30 to 45, from 1 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 1 to 15, from 5 to 15, or from 10 to 15 minutes.

[0128] In some embodiments, amplification can comprise thermocycling of the sample. In some cases, one or more resistive heating elements can be used to perform thermocycling. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20- 45ºC. The nucleic acid amplification reaction can be performed at a temperature no greater than 20ºC, 25ºC, 30ºC, 35ºC, 37ºC, 40ºC, 45ºC, 50ºC, 55ºC, 60ºC, or 65ºC. The nucleic acid amplification reaction can be performed at a temperature of at least 20ºC, 25ºC, 30ºC, 35ºC, 37ºC, 40ºC, 45ºC, 50ºC, 55ºC, 60ºC, or 65ºC. In some cases, the nucleic acid amplification reaction is performed at a temperature of from 20ºC to 45ºC, from 25ºC to 40ºC, from 30ºC to 40ºC, or from 35ºC to 40ºC. In some cases, the nucleic acid amplification reaction is performed at a temperature of from 45ºC to 65ºC, from 50ºC to 65ºC, from 55ºC to 65ºC, or from 60ºC to 65ºC. In some cases, the nucleic acid amplification reaction can be performed at a temperature that ranges from about 20 °C to 45 °C, from 25 °C to 45 °C, from 30 °C to 45 °C, from 35 °C to 45 °C, from 40 °C to 45 °C, from 20 °C to 37 °C, from 25 °C to 37 °C, from 30 °C to 37 °C, from 35 °C to 37 °C, from 20 °C to 30 °C, from 25 °C to 30 °C, from 20 °C to 25 °C, or from about 22 °C to 25 °C. In some cases, the nucleic acid amplification reaction can be performed at a temperature that ranges from about 40 °C to 65 °C, from 45 °C to 65 °C, from 50 °C to 65 °C, from 55 °C to 65 °C, from 60 °C to 65 °C, from 40 °C to 60 °C, from 45 °C to 60 °C, from 50 °C to 60 °C, from 55 °C to 60 °C, from 40 °C to 55 °C, from 45 °C to 55 °C, from 50 °C to 55 °C, from 40 °C to 50 °C, or from about 45 °C to 50 °C.Docket No.203477-779601

[0129] The nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45ºC. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 45-65 ºC. The nucleic acid amplification reaction can be performed at a temperature no greater than 20ºC, 25ºC, 30ºC, 35ºC, 37ºC, 40ºC, 45ºC, 50ºC, 55ºC, 60ºC, or 65ºC. The nucleic acid amplification reaction can be performed at a temperature of at least 20ºC, 25ºC, 30ºC, 35ºC, 37ºC, 40ºC, 45ºC, 50ºC, 55ºC, 60ºC, or 65ºC.

[0130] In some embodiments, each of the plurality of reaction mixtures is in a separate container, such as a tube. In some embodiments, each of the plurality of reaction mixtures is in a well of a multi-well plate (e.g., a 96-well plate). In embodiments where reactions are in tubes or well-plates, devices for controlling temperature (e.g., as in a thermocycler) and / or for detecting signals (e.g., as in a spectrophotometer) may be used to perform one or more steps of a method herein. In some embodiments, each of the plurality of reaction mixtures is in a well or chamber of a microfluidic device. The microfluidic device may comprise any suitable number of chambers. In some embodiments, all of the chambers in the microfluidic device are fluidically connected. In some embodiments, a microfluidic device comprises two or more sets of chambers, in which chambers within a set are fluidically connected in sequence, but chambers in different sets may or may not be connected. For example, two or more sets of chambers may each branch off from a common sample interface or sample preparation region. Alternatively, two or more sets of chambers in a single microfluidic device may be unconnected from each other and fed by separate sample interfaces. In some embodiments, the plurality of chambers comprises at least 10, 25, 50, 75, 100, 150, 200, 300, 400, 500, 1000 chambers fluidically connected in sequence. In some embodiments, the plurality of chambers comprises at least 10, 25, 50, or 100 chambers fluidically connected in sequence. In some embodiments, the plurality of chambers comprises at least 50 chambers fluidically connected in sequence. The microfluidic device may comprise or be configured to interface with one or more heaters for controlling temperature of reactions in the chambers, and one or more detectors for detecting a detectable product in the chambers. When separate from the microfluidic device, the one or more heaters and the detector may be on the same or separate devices configured to interface with the microfluidic device. Non-limiting examples of multi-chamber microfluidic devices for use with assays described herein are provided in PCT / US2022 / 034110, which is incorporated herein by reference in its entirety.

[0131] In some embodiments, reagents for one or more reactions (e.g., amplification and / or programmable nuclease based detection) may be pre-loaded in a container (e.g., a tube,Docket No.203477-779601 well, or chamber). Specific containers may contain reagents for detection of specific target nucleic acids. In some embodiments, one or more detection reagents (e.g., a guide nucleic acid and reporter) can be synthesized in arrayed patterns using photolithography. In some embodiments, each container may comprise a reporter and a different guide nucleic acid which is complementary to a different target nucleic acid of a plurality of target nucleic acids. In some embodiments, one or more programmable nuclease, one or more reporter, and / or one or more guide nucleic acid may be lyophilized, vitrified, or dried in the container. In some embodiments, a plurality of containers contain reagents for detecting the same target nucleic acid. In some embodiments, some of a plurality of chambers having the same guide nucleic acid further comprise different predetermined amounts of the target nucleic acid for use in determining a standard curve.

[0132] In some embodiments, one or more guide nucleic acids can be used to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein. The guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein. The guide nucleic acid is complementary to the target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid can be a RNA, DNA, or synthetic nucleic acids. A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. A guide nucleic acid can be a crRNA. Sometimes, a guide nucleic acid may comprise a crRNA and tracrRNA. The guide nucleic acid can bind specifically to the target nucleic acid. In some cases, the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids. The target nucleic acid can be designed and made to provide desired functions. In some cases, the targeting region of a guide nucleic acid is 20 nucleotides in length. The targeting region of the guide nucleic acid can have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some instances, the targeting region of the guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,Docket No.203477-779601 28, 29, or 30 nucleotides in length. In some cases, the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to bind specifically or be specifically hybridizable. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.

[0133] The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest. The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV 18. Often, guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using theDocket No.203477-779601 methods described herein. The tiling, for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling may comprise gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential. Often, a method for detecting a target nucleic acid may comprise contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some detector nucleic acids of a population of detector nucleic acids. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that can be caused by multiple organisms.

[0134] In some embodiments, programmable nucleases can be used to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. A programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. Often, the signal is present prior to detector nucleic acid cleavage and changes upon detector nucleic acid cleavage. Sometimes, the signal is absent prior to detector nucleic acid cleavage and is present upon detector nucleic acid cleavage. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid. The CRISPR-Cas nucleoprotein complex can comprise a Cas protein (also referred to as a Cas nuclease) complexed with a guide nucleic acid, which can also be referred to as CRISPR enzyme. A guide nucleic acid can be a CRISPR RNA (crRNA). Sometimes, a guide nucleic acid may comprise a crRNA and a trans-activating crRNA (tracrRNA).

[0135] The programmable nuclease system used to detect modified target nucleic acids can comprise CRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), Cas proteins, andDocket No.203477-779601 detector nucleic acids.

[0136] Described herein are reagents comprising a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment or portion. A programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence. The programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and degrades non-specifically nucleic acid in its environment. The programmable nuclease has trans cleavage activity once activated. A programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease). A crRNA and Cas protein can form a CRISPR enzyme.

[0137] Several programmable nucleases are consistent with the methods and devices of the present disclosure. For example, CRISPR / Cas enzymes are programmable nucleases used in the methods and systems disclosed herein. CRISPR / Cas enzymes can include any of the known Classes and Types of CRISPR / Cas enzymes. Programmable nucleases disclosed herein include Class 1 CRISPR / Cas enzymes, such as the Type I, Type IV, or Type III CRISPR / Cas enzymes. Programmable nucleases disclosed herein also include the Class 2 CRISPR / Cas enzymes, such as the Type II, Type V, and Type VI CRISPR / Cas enzymes. Preferable programmable nucleases included in the several devices disclosed herein (e.g., a microfluidic device such as a pneumatic valve device or a sliding valve device or a lateral flow assay) and methods of use thereof include a Type V or Type VI CRISPR / Cas enzyme. Non-limiting examples of programmable nucleases are described herein.

[0138] The detection of a target DNA by a DNA-activated programmable RNA nuclease can be connected to a variety of readouts including fluorescence, lateral flow, electrochemistry, or any other readouts described herein. Multiplexing of programmable DNA nuclease, such as a Type V CRISPR-Cas protein, with a DNA-activated programmable RNA nuclease, such as a Type VI protein, with a DNA reporter and an RNA reporter, can enable multiplexed detection of target ssDNAs or a combination of a target dsDNA and a target ssDNA, respectively. Multiplexing of different RNA-activated programmable RNA nucleases that have distinct RNA reporter cleavage preferences can enable additional multiplexing. Methods for the generation of ssDNA for DNA-activated programmable RNA nuclease-based diagnostics can include (1) asymmetric PCR, (2) asymmetric isothermal amplification, such as RPA, LAMP, SDA, etc. (3) NEAR for the production of short ssDNA molecules, and (4) conversion of RNA targets into ssDNA by a reverse transcriptase followed by RNase H digestion. Thus, DNA-activated programmable RNA nuclease detection of target DNA is compatible with the various systems, kits, compositions, reagents, and methods disclosed herein. For example, target ssDNA detection by Cas13a can be employed in a nucleic acid detection device as disclosed herein.Docket No.203477-779601

[0139] In any of the embodiments described herein, the programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid, which can initiate trans cleavage activity. In some cases, the trans cut or trans cleavage can cut and / or release a reporter. In other cases, the trans cut or trans cleavage can produce an analog of a target, which can be directly detected. Trans cleavage activity can be non-specific cleavage of nearby nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal.

[0140] Reporters, which can be referred to interchangeably as reporters or detector nucleic acids, can be used in conjunction with the compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, etc.) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. The reporter can be suspended in solution or immobilized on a surface. For example, the reporter can be immobilized on the surface of a chamber in a device as disclosed herein. In some cases, the reporter can be immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they are held in position by a magnet placed below the chamber. The reporter can be capable of being cleaved by the activated programmable nuclease, thereby generating a detectable signal. In some cases, the detectable signal can be optically detected. In some embodiments, one or more cascade amplification reactions can occur to further amplify the signal before sensing or detection. In any of the embodiments described herein, the reporter can comprise a single stranded detector nucleic acid comprising a detection moiety.

[0141] As used herein, a detector nucleic acid is used interchangeably with reporter or reporter. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the detector nucleic acid is a single-stranded nucleic acid comprising ribonucleotides. The detector nucleic acid can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the detector nucleic acid may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, fromDocket No.203477-779601 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some cases, the detector nucleic acid has only ribonucleotide residues. In some cases, the detector nucleic acid has only deoxyribonucleotide residues. In some cases, the detector nucleic acid may comprise nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the detector nucleic acid may comprise synthetic nucleotides. In some cases, the detector nucleic acid may comprise at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the detector nucleic acid is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length. In some cases, the detector nucleic acid is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length. In some cases, the detector nucleic acid may comprise at least one uracil ribonucleotide. In some cases, the detector nucleic acid may comprise at least two uracil ribonucleotides. Sometimes the detector nucleic acid has only uracil ribonucleotides. In some cases, the detector nucleic acid may comprise at least one adenine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two adenine ribonucleotides. In some cases, the detector nucleic acid has only adenine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one cytosine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two cytosine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one guanine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two guanine ribonucleotides. A detector nucleic acid can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the detector nucleic acid is from 5 to12 nucleotides in length. In some cases, the detector nucleic acid is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the detector nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. For cleavage by a programmable nuclease comprising Cas13, a detector nucleic acid can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Cas12, a detector nucleic acid can be 10 nucleotides in length.

[0142] The single stranded detector nucleic acid can comprise a detection moiety capable of generating a first detectable signal. Sometimes the detector nucleic acid may comprise aDocket No.203477-779601 protein capable of generating a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, a detection moiety is on one side of the cleavage site. Optionally, a quenching moiety is on the other side of the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety. In some cases, the quenching moiety is 5’ to the cleavage site and the detection moiety is 3’ to the cleavage site. In some cases, the detection moiety is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. Sometimes the quenching moiety is at the 5’ terminus of the detector nucleic acid. Sometimes the detection moiety is at the 3’ terminus of the detector nucleic acid. In some cases, the detection moiety is at the 5’ terminus of the detector nucleic acid. In some cases, the quenching moiety is at the 3’ terminus of the detector nucleic acid. In some cases, the single-stranded detector nucleic acid is at least one population of the single-stranded nucleic acid capable of generating a first detectable signal. In some cases, the single-stranded detector nucleic acid is a population of the single stranded nucleic acid capable of generating a first detectable signal. Optionally, there are more than one population of single-stranded detector nucleic acid. In some cases, there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, or greater than 50, or any number spanned by the range of this list of different populations of single-stranded detector nucleic acids capable of generating a detectable signal. In some cases, there are from 2 to 50, from 3 to 40, from 4 to 30, from 5 to 20, or from 6 to 10 different populations of single-stranded detector nucleic acids capable of generating a detectable signal. In some cases there are from 2 to 50, from 5 to 50, from 10 to 50, from 15 to 50, from 20 to 50, from 25 to 50, from 30 to 50, from 35 to 50, from 40 to 50, from 2 to 40, from 5 to 40, from 10 to 40, from 15 to 40, from 20 to 40, from 25 to 40, from 30 to 40, from 35 to 40, from 2 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, from 2 to 20, from 5 to 20, from 10 to 20, from 15 to 20, from 2 to 10, or from 5 to 10 different populations of single-stranded detector nucleic acids capable of generating a detectable signal.

[0143] In some embodiments, target nucleic acid amplicons are detected by immobilized programmable nuclease probes, such as, for example, CRISPR CAS guide RNA probes (referred to as CRISPR probe). Upon a complementary binding event between a target nucleic acid amplicon and a programmable nuclease probe (e.g., an immobilized CRISPR CAS / guide RNA complex) a cutting event will occur that release a reporter that is then detected by a sensor.

[0144] As described above, the single stranded detector nucleic acid can comprise a detection moiety capable of generating a first detectable signal. Sometimes the detector nucleic acid may comprise a protein capable of generating a signal. A signal can be a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electricDocket No.203477-779601 signal. The generation of the detectable signal from the release of the detection moiety indicates that cleavage by the programmable nuclease has occurred and that the sample contains the target nucleic acid. A detection moiety can be any moiety capable of generating a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. A detector nucleic acid, sometimes, is protein-nucleic acid that can generate a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. Often a calorimetric signal is heat produced after cleavage of the detector nucleic acids. Sometimes, a calorimetric signal is heat absorbed after cleavage of the detector nucleic acids. A potentiometric signal, for example, is electrical potential produced after cleavage of the detector nucleic acids. An amperometric signal can be movement of electrons produced after the cleavage of detector nucleic acid. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the detector nucleic acids. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of detector nucleic acids. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the detector nucleic acid.

[0145] Detecting the presence or absence of a target nucleic acid of interest can involve measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The signal can be measured using one or more sensors integrated with the device or operatively coupled to the device. Thus, the detecting steps disclosed herein can involve measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, a signal can be present in the absence of detector nucleic acid cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal can be produced in a microfluidic device, a fluorescence plate reader (e.g., a spectrophotometer), or other benchtop instrument, after contacting a sample with a composition comprising a programmable nuclease.

[0146] In some cases, the detectable signals can be detected and analyzed in various ways. For example, the detectable signals can be detected using an imaging device. The imaging device can include a digital camera, such a digital camera on a mobile device. The mobile device can have a software program or a mobile application that can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. Any suitable detection or measurement deviceDocket No.203477-779601 can be used to detect and / or analyze the colorimetric, fluorescence, amperometric, potentiometric, or electrochemical signals described herein. In some embodiments, the colorimetric, fluorescence, amperometric, potentiometric, or another electrochemical sign can be detected using a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and / or an oscilloscope).

[0147] In certain aspects of this disclosure, multiplexing refers to parallel sensing of multiple target nucleic acid sequences in one sample by multiple probes.

[0148] The devices of the present disclosure can be used for detection and quantitation of one or more target nucleic acids within the sample. The devices of the present disclosure can comprise one or multiple pumps, valves, reservoirs, and chambers for sample preparation, optional amplification of a target nucleic acid within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by a programmable nuclease.

[0149] Methods consistent with the present disclosure include a multiplexing method of assaying for a plurality of target nucleic acids in a sample. A multiplexing method may comprise contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some reporters (e.g., protein-nucleic acids) of a population of reporter molecules (e.g., protein-nucleic acids), wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample.

[0150] Multiplexing can comprise spatial multiplexing wherein multiple different target nucleic acids are detected at the same time, but the reactions are spatially separated. In some cases, the multiple target nucleic acids are detected using the same programmable nuclease, but different guide nucleic acids. The multiple target nucleic acids sometimes are detected using the different programmable nucleases. In the case wherein multiple target nucleic acids are detected using the different programmable nucleases, the method involves using a first programmable nuclease, which upon activation (e.g., after binding of a first guide nucleic acid to a first target), cleaves a nucleic acid of a first reporter and using a second programmable nuclease, which upon activation (e.g., after binding of a second guide nucleic acid to a second target), cleaves a nucleic acid of a second reporter.

[0151] Sometimes, multiplexing can be single reaction multiplexing wherein multiple different target acids are detected in a single reaction volume. Often, at least two differentDocket No.203477-779601 programmable nucleases are used in single reaction multiplexing. For example, multiplexing can be enabled by immobilization of multiple categories of detector nucleic acids within a fluidic system, to enable detection of multiple target nucleic acids within a single fluidic system. Multiplexing allows for detection of multiple target nucleic acids in one kit or system. In some cases, the multiple target nucleic acids comprise different target nucleic acids to a virus, a bacterium, or a pathogen responsible for one disease. In some cases, the multiple target nucleic acids comprise different target nucleic acids associated with a cancer or genetic disorder. Multiplexing for one disease, cancer, or genetic disorder increases at least one of sensitivity, specificity, or accuracy of the assay to detect the presence of the disease in the sample. In some cases, the multiple target nucleic acids comprise target nucleic acids directed to different viruses, bacteria, or pathogens responsible for more than one disease. In some cases, multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes of the same bacteria or pathogen responsible for a disease, for example, for a wild-type genotype of a bacteria or pathogen and for genotype of a bacteria or pathogen comprising a mutation, such as a single nucleotide polymorphism (SNP) that can confer resistance to a treatment, such as antibiotic treatment. Multiplexing, thus, allows for multiplexed detection of multiple genomic alleles. For example, multiplexing may comprise method of assaying comprising a single assay for a microorganism species using a first programmable nuclease and an antibiotic resistance pattern in a microorganism using a second programmable nuclease. Sometimes, multiplexing allows for discrimination between multiple target nucleic acids of different HPV strains, for example, HPV16 and HPV18. In some cases, the multiple target nucleic acids comprise target nucleic acids directed to different cancers or genetic disorders. Often, multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes, for example, for a wild-type genotype and for SNP genotype. Multiplexing for multiple diseases, cancers, or genetic disorders provides the capability to test a panel of diseases from a single sample. For example, multiplexing for multiple diseases can be valuable in a broad panel testing of a new patient or in epidemiological surveys. Often multiplexing is used for identifying bacterial pathogens in sepsis or other diseases associated with multiple pathogens.

[0152] Furthermore, signals from multiplexing can be quantified. For example, a method of quantification for a disease panel may comprise assaying for a plurality of unique target nucleic acids in a plurality of aliquots from a sample, assaying for a control nucleic acid control in a second aliquot of the sample, and quantifying a plurality of signals of the plurality of unique target nucleic acids by measuring signals produced by cleavage of detector nucleic acids compared to the signal produced in the second aliquot. In this context, a unique target nucleicDocket No.203477-779601 acid refers to the sequence of a nucleic acid that has an at least one nucleotide difference from the sequences of the other nucleic acids in the plurality. Multiple copies of each target nucleic acid can be present. For example, a unique target nucleic population can comprise multiple copies of the unique target nucleic acid. Often the plurality of unique target nucleic acids is from a plurality of bacterial pathogens in the sample.

[0153] In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 2 different target nucleic acids, such as in separate partitions of a given sample, which may be assayed in separate reaction mixtures (e.g., separate tubes, wells, or chambers of a microfluidic device. For quantification purposes, a separate standard curve may be prepared for each of the different target nucleic acids. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 3 different target nucleic acids. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 4 different target nucleic acids. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 5 different target nucleic acids. In some cases, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 6, 7, 8, 9, or 10 different target nucleic acids.

[0154] In some cases, multiplexing can comprise detecting multiple targets with a single probe. Alternatively, multiplexing can comprise detecting multiple targets with multiple probes. The multiple probes can be configured to detect a presence of a particular sequence, target nucleic acid, or a plurality of different target sequences or nucleic acids.

[0155] In some embodiments, any of the devices of the present disclosure can comprise a sample interface configured to receive a sample that may comprise at least one gene of interest. The device can further comprise a channel in fluid communication with the sample interface and a detection chamber. In some cases, the detection chamber is configured to receive and contact the sample, or a portion thereof, with at least one programmable nuclease probe disposed on a surface of said detection chamber. The at least one programmable nuclease probe can comprise a guide nucleic acid complexed with a programmable nuclease. In some cases, the programmable nuclease probe may comprise a CRISPR / Cas enzyme. In some cases, the guide nucleic acid may comprise a guide RNA. In some embodiments, the device may comprise a plurality of programmable nuclease probes comprising different guide RNAs.

[0156] In some cases, any of the devices described herein may comprise a physical filter to filter one or more particles from the sample that do not comprise the one or more targets (e.g., a gene of interest). In some cases, the device may comprise a sample preparation chamber. The sample preparation chamber can comprise a lysing agent. The sample preparation chamber can comprise a heating unit configured for heat inactivation. The sample preparation chamber canDocket No.203477-779601 comprise one or more reagents for nucleic acid purification.

[0157] As described elsewhere herein, the signal produced upon cleavage of a target nucleic acid can be associated with a physical, chemical, or electrochemical change or reaction. The signal can comprise an optical signal, a fluorescent or colorimetric signal, a potentiometric or amperometric signal, and / or a piezo-electric signal.

[0158] In any of the embodiments described herein, the device can further comprise a telemedicine unit configured to provide one or more detection results to a computing unit that is remote from the device. In some embodiments, the telemedicine unit provides one or more detection results to a computing unit that is remote to the device through a cloud-based connection. In some embodiments, the telemedicine unit is HIPAA compliant. In some embodiments, the telemedicine unit transmits encrypted data. The computing unit can comprise a mobile device or a computer. The one or more detection results can indicate a presence, absence, or amount of a target nucleic acid of interest in the sample.

[0159] In another aspect, the present disclosure provides a method for target detection and quantitation. The method can comprise contacting a sample with the device of any of the various aspects and embodiments herein, and detecting a presence, absence, or amount of one or more target nucleic acids in said sample. In some cases, the method can comprise generating one or more detection results indicating the presence, absence, or amount of the one or more target nucleic acids in the sample. In some cases, the method can comprise transmitting the one or more detection results to a remote computing unit. The remote computing unit can comprise, for example, a mobile device.

[0160] In some cases, the method can comprise using a physical filter to filter one or more particles from the sample that do not comprise the one or more target genes of interest. In some cases, the method can comprise lysing the sample before detecting the one or more target genes of interest. In some cases, the method can comprise performing heat inactivation on the sample. In some cases, the method can comprise performing enzyme (e.g., PK) inactivation on the sample. In some cases, the method can comprise performing nucleic acid purification on the sample.

[0161] In some cases, the nucleic acid detection devices and instruments described herein can be configured to implement process control procedures to ensure that the sample preparation, target amplification, and target detection processes are performed accurately and as intended.

[0162] The present disclosure provides various devices, systems, instruments, fluidic devices, methods, compositions, and kits for quantitative nucleic acid tests, which may quickly assess the amount of a target nucleic acid that is present in a sample by using a programmable nuclease that can interact with guide nucleic acids and reporters of the systems described hereinDocket No.203477-779601 to generate a detectable signal. For example, disclosed herein are particular microfluidic devices, instruments, and compositions (e.g., programmable nucleases, guide RNAs, reagents for in vitro transcription, amplification, reverse transcription, and reporters, or any combination thereof) for use in said devices or instruments that are particularly well suited to carry out a highly efficient, rapid, and accurate reactions for detecting the amount of a target nucleic acid in a sample (e.g., using a DETECTR reaction). The systems and programmable nucleases disclosed herein can be used as a companion diagnostic with any of the diseases disclosed herein (e.g., RSV, sepsis, flu, COVID-19), or can be used in reagent kits, point-of-care diagnostics, or over-the-counter diagnostics. The systems may be used as a point of care diagnostic or as a lab test for quantification of a target nucleic acid and, thereby, detection of a condition and / or extent of disease progression, etc., for example, in a subject from which the sample was taken. The systems may be used in various sites or locations, such as in laboratories, in hospitals, in physician offices / laboratories (POLs), in clinics, at remotes sites, or at home. Sometimes, the present disclosure provides various devices, systems, fluidic devices, and kits for consumer genetic use or for over-the-counter use. Guide nucleic acids

[0163] Guide nucleic acids are compatible for use in the devices, instruments, systems, and methods described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, microfluidic devices, benchtop fluorescent readers, etc.) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, and reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting presence, absence, or amount of a target nucleic acid in a sample (e.g., in a DETECTR reaction). In some embodiments, a guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein. In some embodiments, a guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment. In some embodiments, a guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein. In general, a guide nucleic acid comprises a sequence that is complementary to at least a portion of the target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid may be a RNA, DNA, orDocket No.203477-779601 synthetic nucleic acids. A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. A guide nucleic acid can be a crRNA. Sometimes, a guide nucleic acid may comprise a crRNA and tracrRNA. The guide nucleic acid can bind specifically to the target nucleic acid. In some cases, the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids. The target nucleic acid can be designed and made to provide desired functions. In some cases, the targeting region of a guide nucleic acid is 20 nucleotides in length. The targeting region of the guide nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some instances, the targeting region of the guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable or bind specifically. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid. The guideDocket No.203477-779601 nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.

[0164] The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest. The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV 18. Often, guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein. The tiling, for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling may comprise gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential. Often, a method for detecting a target nucleic acid may comprise contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some detector nucleic acids of a population of detector nucleic acids. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that may be caused by multiple organisms. Reporter

[0165] Reporters, which can be referred to interchangeably as reporters or detector nucleic acids, described herein are compatible for use in the devices, instruments, systems, and methods described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, microfluidic devices, benchtop fluorescent readers, etc.) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting presence, absence, or amount of a target nucleic acid in a sample (e.g., using aDocket No.203477-779601 DETECTR reaction). Described herein is a reporter comprising a single stranded detector nucleic acid comprising a detection moiety, wherein the reporter is capable of being cleaved by the activated programmable nuclease, thereby generating a first detectable signal. As used herein, a detector nucleic acid is used interchangeably with reporter or reporter. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the detector nucleic acid is a single-stranded nucleic acid comprising ribonucleotides. The detector nucleic acid can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the detector nucleic acid may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non- ribonucleotide residues. In some cases, the detector nucleic acid has only ribonucleotide residues. In some cases, the detector nucleic acid has only deoxyribonucleotide residues. In some cases, the detector nucleic acid may comprise nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the detector nucleic acid may comprise synthetic nucleotides. In some cases, the detector nucleic acid may comprise at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the detector nucleic acid is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length. In some cases, the detector nucleic acid is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length. In some cases, the detector nucleic acid may comprise at least one uracil ribonucleotide. In some cases, the detector nucleic acid may comprise at least two uracil ribonucleotides. Sometimes the detector nucleic acid has only uracil ribonucleotides. In some cases, the detector nucleic acid may comprise at least one adenine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two adenine ribonucleotide. In some cases, the detector nucleic acid has only adenine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one cytosine ribonucleotide. In someDocket No.203477-779601 cases, the detector nucleic acid may comprise at least two cytosine ribonucleotide. In some cases, the detector nucleic acid may comprise at least one guanine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two guanine ribonucleotide. A detector nucleic acid can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the detector nucleic acid is from 5 to l2 nucleotides in length. In some cases, the detector nucleic acid is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the detector nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. For cleavage by a programmable nuclease comprising Casl3, a detector nucleic acid can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Casl2, a detector nucleic acid can be 10 nucleotides in length. Signals

[0166] The devices, systems, fluidic devices, kits, and methods for detecting the presence and amount of a target nucleic acid in a sample described herein may comprise a generation of a signal indicative of the presence or absence of the target nucleic acid in the sample. The generation of a signal indicative of the presence or absence of the target nucleic acid in the sample as described herein is compatible with the methods, systems, instruments, and devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, microfluidic devices, benchtop fluorescent readers, etc.) and may result from the use of compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting presence, absence, or amount of a target nucleic acid in a sample (e.g., using a DETECTR reaction). As disclosed herein, in some embodiments, detecting the presence, absence, or amount of a target nucleic acid of interest involves measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. Alternatively, or in combination, in some embodiments, detecting the presence, absence, or amount of a target nucleic acid of interest involves measuring a signal emitted from a conjugate bound to a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The conjugates may comprise a nanoparticle, a gold nanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescent nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a fluorescent nanoparticle, a liposome, or a dendrimer. The surface of the conjugate may be coated by a conjugate binding molecule that binds to the detection moiety orDocket No.203477-779601 another affinity molecule of the cleaved detector molecule as described herein. Thus, the detecting steps disclosed herein involve indirectly (e.g., via a reporter) measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, a signal may be present in the absence of detector nucleic acid cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal may be produced in a microfluidic device or benchtop instrument after contacting a sample with a composition comprising a programmable nuclease. Buffers

[0167] The reagents described herein can also include buffers, which are compatible with the devices, systems, instruments, fluidic devices, kits, and methods disclosed herein. The buffers described herein are compatible for use in the devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, microfluidic devices, benchtop fluorescent readers, etc.) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting presence, absence, or amount of a target nucleic acid in the sample (e.g., using a DETECTR reaction). These buffers are compatible with the other reagents, samples, and support mediums as described herein for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry. The methods described herein can also include the use of buffers, which are compatible with the methods disclosed herein. For example, a buffer may comprise HEPES, MES, TCEP, EGTA, Tween 20, KC1, MgCl2, glycerol, or any combination thereof. In some instances, a buffer may comprise Tris-HCl pH 8.8, VLB, EGTA, CH3COOH, TCEP, IsoAmp®, (NH4)2SO4, KCl, MgSO4, Tween20, KOAc, MgOAc, BSA, TCEP, or any combination thereof. In some instances the buffer may comprise from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10,5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM HEPES pH 6.8. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 toDocket No.203477-779601 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KC1. In other instances the buffer may comprise 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl2. The buffer can comprise 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30% glycerol. The buffer can comprise from 0% to 30%, from 5% to 30%, from 10% to 30%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 0% to 25%, from 2% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, from 0% to 20%, from 5% to 20%, from 10% to 20%, from 15% to 20%, from 0% to 15%, from 5% to 15%, from 10% to 15%, from 0% to 10%, from 5% to 10%, or from 0% to 5% glycerol. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM Tris-HCl pH 8.8. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KOAc. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM MgOAc. In some instances the buffer may comprise from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10,5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM EGTA. The buffer can comprise from 0% to 30%, from 5% to 30%, from 10% to 30%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 0% to 25%, from 2% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, from 0% to 20%, from 5% to 20%, from 10% to 20%, from 15% to 20%, from 0% to 15%, from 5% to 15%, from 10% to 15%, from 0% to 10%, from 5% to 10%, or from 0% to 5% Tween 20.Docket No.203477-779601 Compositions Comprising One or More Additives for Improving Assay Signals

[0168] In some embodiments, the reagents described herein may include a composition for improving detection signal strength, detection reaction time, detection reaction efficiency, stability, solubility, or the like. In some embodiments, the composition may comprise one or more additives. The one or more additives may, for example, comprise amino acids or derivatives thereof, chaotrpes, chelators, cyclodextrins, inhibitors, ionic liquids, linkers, metals, non detergent sulfobetaines, organic acids, osmolytes, peptides, polyamides, polymers, polyols, polyols and salts, salts, or combinations thereof. In some embodiments, the one or more additives may, for example, comprise one or more of trichloroacetic acid, L-Arginine, L- Glutamic acid, glycine, L-Proline, L-Histidine, beta(β)-Alanine, L-Serine, L-Arginine ethyl ester dihydrochloride, L-Argininamide dihydrochloride, 6-Aminohexanoic acid, Gly-gly peptide, Gly- gly-gly peptide, tryptone, betaine monohydrate, D-(+)-Trehalose dihydrate, Xylitol, D-Sorbitol, sucrose, hydroxyectoine, Trimethylamine N-oxide dihydrate, methyl alpha(α)-D- gluocopyranoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, adipic acid, ethylenediamine dihydrochloride, guanidine hydrochloride, urea, N-methylurea, N-ethylurea, N-methylformamide, hypotauring, TCEP hydrochloride, GSH (L- Glutathione reduced), GSSG (L-Glutathione oxidized), benzaminidine hydrochloride, ethylenediaminetetraacetic acid disodium salt dihydrate, magnesium chloride hexahydrate, calcium chloride dihydrate, cadmium chloride hydrate, cobalt(II) chloride hexahydrate, Non Detergent Sulfobetaine 195 (NDSB-195), NDSB-201, NDSB-211, NDSB-221, NDSB-256, taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, tacsimate pH 7.0, tetraethylammonium bromide, cholin acetate, 1-Ethyl-3-methylimidazolium acetate, 1-Butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, potassium thiocynate, gadolinium(III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium nitrate, DL-malic acid pH 7.0, lithium citrate tribasic tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p- toluenesulfonate, sodium chloride, potassium chloride, sodium phosphate monobasic monohydrate, potassium phosphate dibasic, sodium sulfate decahydrate, lithium chloride, sodium bromide, glycerol, ethylene glycol, polyethylene glycol 200 (PEG-200), PEG 3350, PEG 8000, PEG monomethyl ether 550, PEG monomethyl ether 750, PEG monomethyl ether 1900, formamide, polypropylene glycol P 400, pentaerythritol ethoxylate, 1,2-Propanediol, polyvinylpyrrolidone K 15, 6-O-α-Maltosyl-β-cyclodextrin, (2-Hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, Methyl-β-cyclodextrin, or any combination thereof.

[0169] In some embodiments, the reagents described herein may include a compositionDocket No.203477-779601 for increasing the signal strength of any of the assays described herein. In some embodiments, the composition comprises water and an additive. In some embodiments, the additive may comprise trehalose, xylitol, D-sorbitol, sucrose, and trimethylamine N-oxide dihydrate, or any combination thereof. In some embodiments, the composition further comprises one or more targets, one or more enzymes, one or more reporters, one or more substrates, or any combination thereof as described herein.

[0170] In some embodiments, the reagents described herein may include a composition for reducing the time it takes for the signal of any of the assays described herein to saturate. In some embodiments, the composition comprises an additive. In some embodiments, the additive may comprise betaine monohydrate, acetamide, GABA, L-proline, beta-alanine, 6- aminohexanonic acid, urea, methylurea, ethylurea, hypotaurine, NDSB-256, ammonium acetate, or any combination thereof. In some embodiments, the composition further comprises one or more targets, one or more enzymes, one or more reporters, one or more substrates, or any combination thereof as described herein.

[0171] In some embodiments, the one or more enzymes may comprise a nuclease. In some embodiments, the one or more enzymes may comprise a programmable nuclease. In some embodiments, the one or more enzymes may comprise a Cas12 enzyme. In some embodiments, the one or more enzymes may comprise a Cas14 enzyme. In some embodiments, the one or more enzymes may comprise a CasPhi enzyme. In some embodiments, the one or more enzymes may comprise a Cas13 enzyme. In some embodiments, the one or more enzymes may comprise HRP. In some embodiments, the one or more enzymes may comprise any one or combination of enzymes presented in this disclosure.

[0172] In some embodiments, the one or more reporters may comprise a reporter free in solution. In some embodiments, the one or more reporters may comprise a reporter immobilized on a surface of a support. In some embodiments, the one or more reporters may comprise biotin. In some embodiments, the one or more reporters may comprise a fluorescent moiety. In some embodiments, the one or more reporters may comprise a nucleic acid tether. In some embodiments, the one or more reporters may comprise a linker. In some embodiments, the one or more reporters may comprise any one or combination of reporters presented in this disclosure.

[0173] In some embodiments, the one or more supports may comprise a glassy substance. In some embodiments, the one or more substrates may comprise a polymeric substance. In some embodiments, the one or more substrates may comprise a hydrogel. In some embodiments, the one or more substrates may comprise any one or combination of substrates presented in this disclosure.Docket No.203477-779601 DETECTR assay immobilization

[0174] CRISPR diagnostic reactions are generally performed in solution where the Cas protein-RNA complexes can freely bind target nucleic acids and reporters. However, reactions where all components are in solution can limit the designs of CRISPR diagnostic assays, especially in microfluidic devices. A system where various components of the CRISPR diagnostic reaction are immobilized on a surface enables designs where multiple readouts can be accomplished within a single reaction chamber.

[0175] Described herein are various methods to immobilize CRISPR diagnostic reaction components to the surface of a reaction chamber, well, or other surface (e.g., a surface of a bead). Any of the devices described herein may comprise one or more immobilized detection reagent components (e.g., programmable nuclease, guide nucleic acid, and / or reporter), or component capable of being immobilized at some point during a particular assay (e.g., through the inclusion of a functionality for immobilization to a surface, such as the surface of a bead). In some embodiments, the surface is a bead, which may be present at the beginning of a reaction, or added later to capture one or more components of a reaction (e.g., unreacted reporters comprising an enzyme, a linker, and a functionality for immobilization). The term “bead” is not limited to any particular size or shape. Beads may be uniform or non-uniform, spherical or non- spherical, regular or irregular. In embodiments, beads are magnetic or paramagnetic. The surface of the beads may comprise a reaction partner for the functional moiety for immobilization (e.g., streptavidin coated beads for capture of targets comprising a biotin functionality). In some embodiments, the beads are microparticles or nanoparticles. A variety of beads for use with nucleic acid based assays are commercially available, a non-limiting example of which includes DYNABEADS in various formats. In certain instances, methods include immobilization of programmable nucleases (e.g., Cas proteins or Cas enzymes), reporters, guide nucleic acids (e.g., gRNAs), or a combination of two or all of these. In some embodiments, various CRISPR diagnostic reaction components are modified with biotin. In some embodiments, these biotinylated CRISPR diagnostic reaction components are tested for immobilization on surfaces coated with streptavidin. In some embodiments, the biotin-streptavidin interaction is used as a model system for other immobilization chemistries.Docket No.203477-779601

[0176] Table 2 presents gRNA and reporter immobilization sequences Table 2: gRNA and reporter immobilization sequencesDocket No.203477-779601Docket No.203477-779601Docket No.203477-779601

[0177] FIGS.7A-7C illustrate three exemplary immobilization strategies for CRISPR- Cas diagnostic assay components. In some embodiments, as seen in FIG.7A, chemical modifications of amino acid residues in the Cas protein enable attachment to a surface. In some embodiments, as seen in FIG.7B, gRNAs are immobilized by adding various chemical modifications at the 5’ or 3’ end of the gRNA that are compatible with a selected surface chemistry. In some embodiments, as seen in FIG.7C fluorescence-quenching (FQ), or other reporter chemistries, are attached to surfaces using similar chemical modifications as gRNAs. In some embodiments, these attached reporters are activated by a Cas protein, which leads to eitherDocket No.203477-779601 activated molecules that remain attached to the surface or activated molecules that are released into solution.

[0178] For some embodiments, described herein, FIG.8 provides an illustrative example of immobilization strategies for use with methods and compositions described herein where the RNP complex is immobilized by a gRNA and cleaves surrounding FQ reporters that are also immobilized to a surface. Here, the quencher is released into solution, leaving a localized fluorescent signal.

[0179] In some embodiments, the programmable nuclease, guide nucleic acid, or the reporter are immobilized to a device or well surface by a linkage or linker. In some embodiments, the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, a bond between streptavidin and biotin, an amide bond, or any combination thereof. In some embodiments, the linkage comprises non-specific absorption. In some embodiments, the programmable nuclease is immobilized to the device or well surface by the linkage, wherein the linkage is between the programmable nuclease and the surface. In some embodiments, the reporter is immobilized to the device or well surface by the linkage, wherein the linkage is between the reporter and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 5’ end of the guide nucleic acid and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 3’ end of the guide nucleic acid and the surface. Sample Preparation and Lyophilization

[0180] Described herein are various methods of sample preparation and reagent storage. Any of the devices, systems, or compositions described herein may comprise one or more sample preparation reagents Any of the devices, systems, or compositions described herein may comprise sample preparation reagents as dried reagents. Dried reagents may comprise solids and / or semi-solids. In certain instances, dried reagents may comprise lyophilized reagents. Any of the devices described herein may comprise one or more lyophilized reagents (e.g., amplification reagents, programmable nucleases, buffers, excipients, etc.). In certain instances, methods include sample lysis, concentration, and / or filtration. In certain instances, methods include reconstitution of one or more lyophilized reagents. In some embodiments, lyophilized reagents may be in the form of lyophilized beads, spheres, and / or particulates. In some embodiments, the lyophilized bead, sphere, and / or particulate may comprise either single or multiple compounds. In some embodiments, the lyophilized bead, sphere, and / or particulate may be adjusted to various moisture levels or hygroscopy. In some embodiments, the lyophilizedDocket No.203477-779601 bead, sphere, and / or particulate may comprise assay internal standards. In some embodiments, the lyophilized bead, sphere, and / or particulate may have diameters between about 0.5 millimeters to about 5 millimeters in diameter.

[0181] Described herein are various embodiments of the DETECTR reaction involving optimization of sample preparation and lyophilization. Such embodiments allow for adapting the buffer for binding a substrate to perform a concentration step. In some embodiments, experiments may be performed to evaluate the lysis (sample is evaluated directly in the assay) and binding (the sample is eluted from magnetic beads) characteristics of buffers with different components. In such embodiments, the input sample is the same concentration as the eluted sample. One-pot sRCA: Rolling circle amplification with Cas14a DETECTR in single reaction volume

[0182] Described herein are various methods of sample amplification and detection in a single reaction volume. Any of the devices described herein may be configured to perform amplification and detection in a same well, chamber, channel, or volume in the device. In certain instances, methods include simultaneous amplification and detection in the same volume. In some embodiments, sample amplification may comprise RCA.

[0183] FIG.9 presents an overview of sRCA. In this system, a target nucleic acid is added to a system that contains components for a one-pot RCA + Cas protein reaction. The RCA portion of the system is composed of a dumbbell-shaped DNA template, a primer, and a DNA polymerase. For some embodiments, the DETECTR portion of the reaction is composed of a Cas protein, such as Cas12 or Cas14, a crRNA that targets the amplicon generated by RCA, but not the dumbbell-shaped DNA template, and a FQ ssDNA reporter. A target nucleic acid (e.g., viral RNA), which is capable of binding to the dumbbell DNA template, is added to the system. The target nucleic acid base pairs with the DNA template. The more extensive base pairing between the target and the DNA template causes the internal base pairing of the dumbbell to be disrupted, which opens up a binding site for the primer. The DNA polymerase can then use this primer to begin RCA. In some embodiments, As RCA proceeds, the amplicon is generated which contains the target site for the Cas protein. The Cas protein recognizes this site through base pairing with the crRNA and initiates trans-cleavage of the FQ ssDNA reporter. In some embodiments, the system contains fewer components than other one-pot approaches and does not require a RT enzyme.Docket No.203477-779601 CasPin: Cas13 positive-feedback loop leveraging Cas13 ssDNA targeting

[0184] Described herein are various methods of signal amplification. Any of the devices or systems described herein may be configured to perform signal amplification after the reporter has been cleaved by the programmable nuclease. Signal amplification may improve detection of rare targets in a complex sample. In certain instances, methods include leveraging ssDNA targeting of the programmable nuclease (e.g., Cas13) to create a positive feedback loop upon biding of the programmable nuclease to the target nucleic acid to cleave additional reporters and amplify the signal generated by the presence of the target nucleic acid.

[0185] FIG.10 presents an overview of an RCA-based positive feedback system for a Cas13 effector protein. In some embodiments, Cas13 may be programmed to recognize an RNA target. When the RNA target is present, a blocking motif on the 3’ end of a primer is removed. After removal of this blocking motif, the primer can then serve to open up the circular template and allow for amplification by RCA using a DNA polymerase. As the amplicon is generated the same target sequence as the original RNA is generated. This ssDNA target sequence is then capable of being recognized by Cas13 which can either remove additional blocking groups from primers or cleave a reporter that generates a fluorescent signal. This system forms a positive feedback loop for Cas13. In this context, a “blocking motif” refers to a moiety at the 3’ end of a polynucleotide that impedes formation of a covalent bond between the 3′ hydroxyl moiety of the nucleotide and the 5′ phosphate of another nucleotide. A variety of suitable blocking motifs are available, non-limiting examples of which are described in US20210198730, which is incorporated herein by reference.

[0186] FIG.11 presents an overview of CasPin. In some embodiments, the CasPin system uses two populations of Cas13. One is programmed with a crRNA that targets an RNA of interest, such as a viral genome. The other population is programmed with a crRNA that is optimal for ssDNA detection. In some embodiments, the systems also contains a hairpin-shaped oligo that is composed of both DNA and RNA. Finally, in some embodiments, there is a FQ RNA reporter that is used to readout the result of the assay. When Cas13 detects the RNA of interest, it can either cleave the FQ RNA reporter or the RNA on the hairpin oligo. When the RNA on the hairpin oligo is cleaved, it dissociates from the DNA revealing a ssDNA target site that can be recognized by the other population of Cas13 RNPs. This initiates a positive feedback loop where Cas13 recognizes the ssDNA target and cleaves more hairpin molecules, which increases the overall amount of target in the system, and leads to further activation of the system. As this process continues more and more FQ RNA reporter is cleaved, which is the ultimate readout of the assay.Docket No.203477-779601

[0187] FIG.12 presents potential structures of hairpins for CasPin. In some embodiments, the target ssDNA sequence is indicated by the purple rectangle. RNA loop structures could occur on either side of the target strand (either 5’, 3’ or both). The strand that is complementary to the target site could be DNA or RNA. The strand that is complementary to the target site could also be a perfect match to the target site, be shorter than the target site, or contain mismatches to help destabilize or promote trans-cleavage by Cas13.

[0188] In some embodiments, two hairpins are used on either end of the target site. FIG. 13 presents results for such an embodiment and indicates capability for blocking Cas13 from recognizing the ssDNA target site. In some embodiments, CasPin oligos have varying lengths of hairpin stems. In some embodiments, CasPin oligo do not have stem structures. In some embodiments, CasPin oligos contain another DNA sequence. Such embodiments were evaluated and found to not be recognized by the crRNA. Both raw oligos from the manufacture and those that had been denatured and refolded at 25C in a Cas13 DETECTR reaction were tested. The results of this experiment demonstrated that Cas13 was able to recognize the target site regardless of the stem length. In some embodiments, longer stem length oligos block Cas13 recognition without RNA cleavage to release the structure. One-pot DETECTR on handheld microfluidic device

[0189] Described herein are various devices and methods for running one-pot DETECTR assays on a handheld device. Any of the devices described herein may be configured to perform a one-pot DETECTR assay. DETECTR based multiplexed detection

[0190] FIG.1 shows a schematic of an exemplary workflow for a multiplexed programmable nuclease assay. Any of the devices or systems described herein may be configured to perform one or more of the reactions described herein (e.g., amplification, detection, etc.) in separate chambers or wells (e.g., in a microfluidic device, or on a microwell plate). In at least some instances, fluidly isolating reactions for different target nucleic acids may facilitate multiplexing.

[0191] In some embodiments, each reaction chamber or well may comprise one or more guide nucleic acids (e.g., sgRNAs). In some embodiments, the one or more guide nucleic acids may be immobilized to a surface (e.g., a glass bead or hydrogel disposed within a reaction chamber or well). In some embodiments, the guide nucleic acids are part of a programmable nuclease (e.g., Cas-complex) probe immobilized to a surface. In some embodiments, a guide nucleic acid is designed to specifically bind to a target nucleic acid in the sample. In someDocket No.203477-779601 embodiments, there are different guide nucleic acids corresponding to different reaction chambers, where each different guide nucleic acid is complimentary for a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing guide nucleic acids, each reaction chamber may contain or be functionalized with one or more reporters having distinct functional groups as described herein. In some embodiments, the reporters may be in close enough proximity to be cleaved by the programmable nuclease probes. In some embodiments, reporters are cleaved and portion thereof (e.g., a detection moiety) is released into the solution upon binding between a particular guide nucleic acid and the target nucleic acid to which the guide nucleic acid is designed to specifically bind. In some embodiments, reporters are functionalized with a detection moiety (e.g., a label). Nicking Enzyme Amplification Reaction (NEAR)

[0192] In some embodiments, a target nucleic acid may be amplified using a nicking enzyme amplification reaction (NEAR). NEAR may be used to amplify a region of a nucleic acid comprising a target nucleic acid. NEAR may comprise a forward primer and a reverse primer that at least partially anneals to complementary strands of a target nucleic acid 3’ of the region to be amplified. The forward primer and the reverse primer may comprise a stabilizing region that is not complementary to the target sequence. The forward primer and the reverse primer may comprise recognition regions that may be nicked by a nicking enzyme. A polymerase may polymerize a nucleic acid 5’ to 3’ from the 3’ end of the forward primer or the 3’ end of the reverse primer, using the strand to which the primer is annealed as a template, resulting in a double stranded nucleic acid product or amplicon. The newly synthesized strand may comprise a restriction site and may then serve as a template. The resulting double stranded nucleic acid amplicon may comprise nicking sites in both strands. A nicking enzyme may nick a single strand of the double stranded product or amplicon. The polymerase may polymerize a nucleic acid 5’ to 3’ from the 3’ end of the nucleic acid amplicon 5’ of the nick. The process may be repeated, thereby amplifying the target nucleic acid.

[0193] As described herein, a target nucleic acid may be detected using a DNA-activated programmable RNA nuclease (e.g., a Cas13), a DNA-activated programmable DNA nuclease (e.g., a Cas12), or an RNA-activated programmable RNA nuclease (e.g., a Cas13) and other reagents disclosed herein (e.g., RNA components). The target nucleic acid may be detected using DETECTR, as described herein. The target nucleic acid may be an RNA, reverse transcribed RNA, DNA, DNA amplicon, amplified DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. In some embodiments, the target nucleic acid is amplifiedDocket No.203477-779601 prior to or concurrent with detection. In some embodiments, the target nucleic acid is reverse transcribed prior to amplification. The target nucleic acid may be amplified via NEAR of a target nucleic acid sequence. In some embodiments, the nucleic acid is amplified using NEAR coupled with reverse transcription (RT-NEAR). The NEAR amplification may be performed independently, or the NEAR amplification may be coupled to DETECTR for detection of the target nucleic acid. The RT-NEAR amplification may be performed independently, or the RT- NEAR amplification may be coupled to DETECTR for detection of the target nucleic acid. The DETECTR reaction may be performed using any method consistent with the methods disclosed herein.

[0194] NEAR may be performed as an isothermal reaction, for example NEAR may be performed at from about 37 °C to about 42 °C. In some embodiments, NEAR may be performed at about room temperature. In some embodiments, a nucleic acid sample may be heated prior to isothermal amplification. In some embodiments, the nucleic acid sample heated prior to isothermal amplification may comprise one or more primers. The nucleic acid sample may be heated to about 95 °C prior to isothermal amplification. The nucleic acid sample may be heated to a temperature sufficient to dissociate two strands of a double stranded nucleic acid sequence.

[0195] NEAR may amplify a target nucleic acid to detectable levels within about 30 seconds, 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes.

[0196] NEAR Amplification and Detection Reaction Mixtures

[0197] NEAR reaction components may comprise a polymerase, a nicking enzyme, dNTPs, and one or more nucleic acid primers. In some embodiments, the reaction may further comprise a reverse transcriptase as described herein. In some embodiments, the polymerase may be an exo-Klenow polymerase. The nicking enzyme may be capable of nicking a single strand of a double stranded nucleic acid sequence. In some embodiments, the nicking enzyme may be capable of nicking an unthiolated strand of a double stranded nucleic acid sequence comprising a thiolated strand and an unthiolated strand. In some embodiments, the nicking enzyme may be capable of nicking a single strand comprising an unthiolated region of a double stranded nucleic acid comprising at one or more thiolated regions and one or more unthiolated regions. In some embodiments, the nicking enzyme is a restriction enzyme capable of nicking a single strand of a double stranded nucleic acid sequence. In some embodiments, the nicking enzyme is a modified restriction enzyme. The nicking enzyme may be a strand-limited restriction enzyme. The restriction enzyme may be HincII. In some embodiments, the restriction enzyme may be AluI, BamHI, EcoP15I, EcoRI, EcoRII, EcoRV, HaeIII, HgaI, HindII, HindIII, HinFI, KpnI, NotI,Docket No.203477-779601 PstI, PvuII, SacI, SalI, Sau3, ScaI, SmaI, SpeI, SphI, StuI, TaqI, or XbaI, or the like. The nicking enzyme may be Nt.BspQI, Nt.CvPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.BsmAI, Nb.Bpu10I, Nt.Bpu110I, Nb.Mva1269I, or I-HmuI, or the like. The one or more nucleic acid primers may comprise two primers. For example, the one or more nucleic acid primers may comprise a first primer (e.g., an S1 primer) and a second primer (e.g., an S2 primer). The target nucleic acid may be single stranded DNA or double stranded DNA. In some embodiments, a target nucleic acid comprising RNA may be reverse transcribed into DNA using a reverse transcriptase prior to NEAR amplification. A reverse transcription reaction may comprise primers, dNTPs, and a reverse transcriptase. In some embodiments, the reverse transcription reaction and the NEAR amplification reaction may be performed in the same reaction. A combined RT-NEAR reaction may comprise NEAR primers, reverse transcription primers, dNTPs, a reverse transcriptase, a polymerase, and dNTPs. In some embodiment, the NEAR primers may comprise the reverse transcription primers.

[0198] A DETECTR reaction to detect the target nucleic acid sequence may comprise a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease. The programmable nuclease when activated, as described elsewhere herein, exhibits sequence-independent cleavage of a reporter (e.g., a nucleic acid comprising a moiety that becomes detectable upon cleavage of the nucleic acid by the programmable nuclease). The programmable nuclease is activated upon the guide nucleic acid hybridizing to the target nucleic acid. A combined NEAR DETECTR reaction may comprise a polymerase, a restriction enzyme, dNTPs, one or more nucleic acid primers, a guide nucleic acid, a programmable nuclease, and a substrate nucleic acid. A combined RT-NEAR DETECTR reaction may comprise reverse transcription primers, a reverse transcriptase, a polymerase, a restriction enzyme, dNTPs, one or more nucleic acid primers, a guide nucleic acid, a programmable nuclease, and a substrate nucleic acid. In some embodiment, the primers may comprise the reverse transcription primers. NEAR and DETECTR can be carried out in the same sample volume. NEAR and DETECTR can be carried out concurrently in the same sample volume. RT-NEAR and DETECTR can be carried out in the same sample volume. RT-NEAR and DETECTR can be carried out concurrently in the same sample volume. A NEAR reaction may be multiplexed to amplify a plurality of target nucleic acid sequences in a single reaction.

[0199] NEAR Primers and Guide Nucleic Acids

[0200] A number of NEAR primers and NEAR primer design methods are consistent with the methods compositions, reagents, enzymes, and kits disclosed herein. NEAR may comprise a set of primers. In some embodiments, NEAR may be an RT-NEAR reaction, a NEAR DETECTR reaction, or an RT-NEAR DETECTR reaction. The set of NEAR primersDocket No.203477-779601 may comprise two primers, a first primer and a second primer. In some embodiments, a first primer may comprise a sequence of the first region at the 3’ end of the first primer. The sequence of the first region may be from about 16 nucleic acids to about 25 nucleic acids long, or about 20 nucleic acids long. The 3’ end of the first primer may hybridize to the first complementary region of the target. The first complementary region may be 3’ of the target nucleic acid. The first complementary region may be 3’ of a sequence reverse complementary to the target nucleic acid. The first primer may further comprise a cut site 5’ of the sequence of the first region that may be recognized and cleaved by a nicking enzyme. The 3’ end of the first primer may further comprise a recognition site for a nicking enzyme. In some embodiments, the 3’ end of the first primer may further comprise a nicking enzyme stabilization region. The sequence of the second region may be from about 30 nucleic acids to about 38 nucleic acids long. The 3’ end of the second primer may hybridize to the second complementary region. The second complementary region may be 3’ of a sequence reverse complementary to the target nucleic acid. The second complementary region may be 3’ of the target nucleic acid. The second complementary region may be 3’ of a sequence reverse complementary to the target nucleic acid. The second primer may further comprise a cut site 5’ of the sequence of the second region that may be recognized and cleaved by a nicking enzyme. The 3’ end of the second primer may further comprise a recognition site for a nicking enzyme. In some embodiments, the 3’ end of the second primer may further comprise a nicking enzyme stabilization region.

[0201] The NEAR primers are designed depending on the site of the optimal guide RNA placement, which may or may not be determined by an available PAM sequence. When performing a NEAR-DETECTR reaction, single-stranded DNA is produced by the designed primers. Because the DETECTR reaction will detection single stranded DNA species, the amplification reaction can be biased to produce more of the particular strand than another. This can be done through changing of the ratio of the forward and reverse primer concentrations. In some embodiments, the concentration of forward primer can be 5 times, 4 times, 3 times, 2 times, or equal to the concentration of reverse primer. In some embodiments, the concentration of reverse primer can be 5 times, 4 times, 3 times, 2 times, or equal to the concentration of forward primer.

[0202] In some embodiments, the first region, the second region, or both may be about 8 nucleic acids, about 10 nucleic acids, about 12 nucleic acids, about 14 nucleic acids, about 16 nucleic acids, about 18 nucleic acids, about 20 nucleic acids, about 22 nucleic acids, about 24 nucleic acids, about 26 nucleic acids, about 28 nucleic acids, about 30 nucleic acids, about 32 nucleic acids, about 34 nucleic acids, about 36 nucleic acids, about 38 nucleic acids, about 40 nucleic acids, about 42 nucleic acids, about 44 nucleic acids, about 46 nucleic acids, about 48Docket No.203477-779601 nucleic acids, or about 50 nucleic acids long.

[0203] In some embodiments, the first region, the second region, or both may be from about 8 to about 12, from about 8 to about 16, from about 8 to about 20, from about 8 to about 24, from about 8 to about 28, from about 8 to about 30, from about 8 to about 32, from about 8 to about 34, from about 8 to about 36, from about 8 to about 38, from about 8 to about 40, from about 8 to about 42, from about 8 to about 44, from about 8 to about 48, or from about 8 to about 50 nucleic acids long.

[0204] In some embodiments, the first region, the second region, or both may comprise a GC content of about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In some embodiments, the first region, the second region, or both may comprise a GC content of from about 1% to about 95%, from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, from about 20% to about 60%, from about 25% to about 50%, or from about 30% to about 40%

[0205] In some embodiments, the first region, the second region, or both may have a melting temperature of about 38 °C, about 40 °C, about 42 °C, about 44 °C, about 46 °C, about 48 °C, about 50 °C, about 52 °C, about 54 °C, about 56 °C, about 58 °C, about 60 °C, about 62 °C, about 64 °C, about 66 °C, about 68 °C, about 70 °C, about 72 °C, about 74 °C, about 76 °C, about 78 °C, about 80 °C, about 82 °C, about 84 °C, about 86 °C, about 88 °C, about 90 °C, or about 92 °C. In some embodiments, the first region, the second region, or both may have a melting temperature of from about 35 °C to about 40 °C, from about 35 °C to about 45 °C, from about 35 °C to about 50 °C, from about 35 °C to about 55 °C, from about 35 °C to about 60 °C, from about 35 °C to about 65 °C, from about 35 °C to about 70 °C, from about 35 °C to about 75 °C, from about 35 °C to about 80 °C, or from about 35 °C to about 85 °C.

[0206] A set of NEAR primers may be designed for use in combination with a DETECTR reaction. The amplified nucleic acid sequence may comprise a sequence that hybridizes to a guide RNA. The amplified nucleic acid sequence may comprise a target nucleic acid. The guide RNA may hybridize to the target nucleic acid. The amplified nucleic acid sequence may comprise corresponding to a guide RNA. The amplified nucleic acid sequence may comprise a sequence reverse complementary to the target nucleic acid. All or part of the guide RNA may be reverse complementary to all or part of the target nucleic acid. The amplified nucleic acid sequence may comprise a protospacer adjacent motif (PAM) positioned next to or near the target sequence. The PAM may be 3’ of the target nucleic acid. In some embodiments, a portion of a sequence that hybridizes the guide RNA may be located between the first region and the second complementary region. The portion of a sequence that hybridizes the guide RNADocket No.203477-779601 located between the first region and the second complementary region may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more of the sequence that hybridizes guide RNA. In some embodiments, the 5’ end of the sequence that hybridizes the guide RNA is 3’ of the 3’ end of the first region and 3’ end of the sequence that hybridizes the guide RNA is 5’ of the 5’ end of the second complementary region. In some embodiments, a portion of a sequence that hybridizes the guide RNA may be located between the second region and the first complementary region. The portion of a sequence that hybridizes the guide RNA located between the second region and the first complementary region may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more of the sequence that hybridizes the guide RNA. In some embodiments, the 5’ end of the sequence that hybridizes the guide RNA is 3’ of the 3’ end of the second region and 3’ end of the sequence that hybridizes the guide RNA is 5’ of the 5’ end of the first complementary region.

[0207] In some embodiments, a sequence that hybridizes the guide RNA may overlap the first region, the first complementary region, the second region, or the second complementary region by no more than no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 35%, no more than about 40%, no more than about 45%, no more than about 50%, no more than about 55%, no more than about 60%, no more than about 65%, no more than about 70%, no more than about 75%, no more than about 80%, no more than about 85%, no more than about 90%, or no more than about 95%. In some embodiments, the sequence that hybridizes the guide RNA does not overlap the first region, the first complementary region, the second region, or the second complementary region. In some embodiments, the guide RNA does not hybridize to the first primer or the second primer.

[0208] In some embodiments, a NEAR primer set may be designed using a commercially available primer design software. A NEAR primer set may be designed for use in combination with a DETECR reaction, a reverse transcription reaction, or both. One or more methods of designing a set of NEAR primers may be readily apparent to one skilled in the art and may be employed in any of the compositions, kits and methods described herein.Docket No.203477-779601 TABLE 3 – Exemplary NEAR PrimersDocket No.203477-779601 Amplification and Detection of a Gene of Interest Comprising a SNP

[0209] A DETECTR reaction may be used to detect the presence of a specific target gene in the same. The DETECTR reaction may produce a detectable signal, as described elsewhere herein, in the presence of a target nucleic acid sequence comprising a target gene. The DETECTR reaction may not produce a signal in the absence of the target nucleic acid or in the presence of a nucleic acid sequence that does not comprise the specific SNP allele or comprises a different SNP allele or the target gene. In some embodiments, a DETECTR reaction may comprise a guide RNA reverse complementary to a portion of a target nucleic acid sequence comprising a specific SNP allele. The guide RNA and the target nucleic acid comprising the specific SNP allele may bind to and activate a programmable nuclease, thereby producing a detectable signal as described elsewhere herein. The guide RNA and a nucleic acid sequence that does not comprise the specific SNP allele may not bind to or activate the programmable nuclease and may not produce a detectable signal. In some embodiments, a target nucleic acid sequence that may or may not comprise a specific SNP allele may be amplified using, for example, a LAMP amplification reaction, an RPA amplification reaction, an SDA amplification reaction, a NEAR amplification reaction, or any other amplification method. In some embodiments, the amplification reaction may be combined with a reverse transcription reaction, a DETECTR reaction, or both. For example, the amplification reaction may be an RT-NEAR reaction, a NEAR DETECTR reaction, or an RT-NEAR DETECTR reaction. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.

[0210] A DETECTR reaction, as described elsewhere herein, may produce a detectable signal specifically in the presence of a target nucleic acid sequence comprising a target gene. In addition to the DETECTR reaction, the target nucleic acid having the target gene may be amplified. For example, the reactions can comprise NEAR and DETECTR reactions, or RT- NEAR and DETECTR reactions. Performing a DETECTR reaction in combination with a NEAR reaction may result in an increased detectable signal as compared to the DETECTR reaction in the absence of the NEAR reaction. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.

[0211] In some embodiments, the detectable signal produced in the DETECTR reaction may be higher in the presence of a target nucleic acid comprising target nucleic acid than in the presence of a nucleic acid that does not comprise the target nucleic acid. In some embodiments, the DETECTR reaction may produce a detectable signal that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, atDocket No.203477-779601 least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at last 400- fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold greater in the presence of a target nucleic acid comprising a target nucleic acid than in the presence of a nucleic acid that does not comprise the target nucleic acid. In some embodiments, the DETECTR reaction may produce a detectable signal that is from 1-fold to 2- fold, from 2-fold to 3-fold, from 3-fold to 4-fold, from 4-fold to 5-fold, from 5-fold to 10-fold, from 10-fold to 20-fold, from 20-fold to 30-fold, from 30-fold to 40-fold, from 40-fold to 50- fold, from 50-fold to 100-fold, from 100-fold to 500-fold, from 500-fold to 1000-fold, from 1000-fold to 10,000-fold, from 10,000-fold to 100,000-fold, or from 100,000-fold to 1,000,000- fold greater in the presence of a target nucleic acid comprising a specific SNP allele than in the presence of a nucleic acid that does not comprise the specific SNP allele. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.

[0212] A DETECTR reaction may be used to detect the presence of a target nucleic acid associated with a disease or a condition in a nucleic acid sample. The DETECTR reaction may reach signal saturation within about 30 seconds, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 75 minutes, about 80 minutes, or about 85 minutes and be used to detect the presence of a target gene associated with an increased likelihood of developing a disease or a condition in a nucleic acid sample. The DETECTR reaction may be used to detect the presence of a target gene associated with a phenotype in a nucleic acid sample. For example, a DETECTR reaction may be used to detect target gene associated with a disease such as phenylketonuria (PKU), cystic fibrosis, sickle-cell anemia, albinism, Huntington's disease, myotonic dystrophy type 1, hypercholesterolemia, neurofibromatosis, polycystic kidney disease, hemophilia, muscular dystrophy, hypophosphatemic rickets, Rett's syndrome, or spermatogenic failure. A DETECTR reaction may be used to detect a SNP allele associated with an increased risk of cancer, for example bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, gallbladder cancer, stomach cancer, leukemia, liver cancer, lung cancer, oral cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, thyroid cancer, neuroblastoma, or lymphoma. A DETECTR reaction may be used to detect a SNP allele associated with an increased risk of a disease, for example Alzheimer’s disease, Parkinson’s disease, amyloidosis, heterochromatosis, celiac disease,Docket No.203477-779601 macular degeneration, or hypercholesterolemia. A DETECTR reaction may be used to detect a SNP allele associated with a phenotype, for example, eye color, hair color, height, skin color, race, alcohol flush reaction, caffeine consumption, deep sleep, genetic weight, lactose intolerance, muscle composition, saturated fat and weight, or sleep movement. A DETECTR reaction may also be used to detect the presence of a pathological organism. In some embodiments, the pathological organism is a prokaryote, eukaryote, or a protozoa. In some embodiments, the pathological organism is a virus, an opportunistic pathogen, a parasite, a bacterium, or any combination thereof. In some embodiments, the pathological organism is SARS-CoV-2 or Streptococcus pyogenes.

[0213] The terms “sample interface”, “sample input”, “input port”, “input”, “port” as used herein, generally refers to a portion of a device that is configured to receive a sample.

[0214] The terms “heating region”, “heated region”, “heat chambers”, “heat volumes”, “heat zones”, “heat surfaces”, “heat areas”, and the like, as used herein, generally refers to a portion of a device that is in thermal communication with a heating unit.

[0215] The terms “heater”, “heating unit”, “heating element”, “heat source”, and the like, as used herein, generally refers to an element that is configured to produce heat and is in thermal communication with a portion of a device.

[0216] The term “reagent mix”, “reagent master mix”, “reagents”, and the like, as used herein, generally refers to a formulation comprising one or more chemicals that partake in a reaction that the reagent mix is formulated for.

[0217] The term “non-cycled temperature profile,” as used herein, generally refers to a temperature profile that is cyclical or sinusoidal in that the temperature profile has an initial temperature, a target temperature, and a final temperature.

[0218] The term “collection tube,” as used herein, generally refers to a compartment that is used to collect a sample and deliver the sample to the sample interface of a device. In some embodiments, the collection tube may be portable. In some embodiments, the collection tube is a syringe.

[0219] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), or + / - 10% of the stated value (or rangeDocket No.203477-779601 of values). Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points.

[0220] As used herein, the terms “thermostable” and “thermostability” refer to the stability of a composition disclosed herein at one or more temperatures, such as an elevated operating temperature for a given reaction. Stability may be assessed by the ability of the composition to perform an activity, e.g., cleaving a target nucleic acid or reporter. Improving thermostability means improving the quantity or quality of the activity at one or more temperatures.

[0221] As used herein, the terms “percent identity,” “% identity,” and “% identical” refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y in an alignment between the two. Generally, computer programs may be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci.1988 Mar;4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A.1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res.1997 Sep 1;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res.1984 Jan 11;12(1 Pt 1):387-95). For the purposes of calculating identity to the sequence, extensions, such as tags, are not included.

[0222] As used herein, a “one-pot” reaction refers to a reaction in which more than one reaction occurs in a single volume alongside a programmable nuclease-based detection (e.g., DETECTR) assay. For example, in a one-pot assay, sample preparation, reverse transcription, amplification, in vitro transcription, or any combination thereof, and programmable nuclease- based detection (e.g., DETECTR) assays (optionally including signal amplification) are carried out in a single volume. In some embodiments, amplification and detection are carried out within a same volume or region of a device (e.g., within a detection region). Readout of the detection (e.g., DETECTR) assay may occur in the single volume or in a second volume. For example, the product of the one-pot DETECTR reaction (e.g., a cleaved detection moiety comprising an enzyme) may be transferred to another volume (e.g., a volume comprising an enzyme substrate)Docket No.203477-779601 for signal generation and indirect detection of reporter cleavage by a sensor or detector (or by eye in the case of a colorimetric signal).

[0223] As used herein, “HotPot” refers to a one-pot reaction in which both amplification (e.g., RT-LAMP) and detection (e.g., DETECTR) reactions occur simultaneously. In many embodiments, a HotPot reaction may utilize a thermostable programmable nuclease which exhibits trans cleavage at elevated temperatures (e.g., greater than 37C).

[0224] The terms, “nucleic acid amplification” and “amplifying a nucleic acid,” as used herein, refer to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof.

[0225] The term, “complementary,” as used herein with reference to a nucleic acid refers to the characteristic of a polynucleotide having nucleotides that base pair with their Watson- Crick counterparts (C with G; or A with T / U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5′- to 3′-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3′- to its 5′-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5′- to its 3′-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[0226] The term, “cleavage assay,” as used herein refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid. In some cases, the cleavage activity may be cis-cleavage activity. In some cases, the cleavage activity may be trans-cleavage activity.

[0227] Assays which leverage the transcollateral cleavage properties of programmable nuclease enzymes (e.g., CRISPR-Cas enzymes) are often referred to herein as DNA endonuclease targeted CRISPR trans reporter (DETECTR) reactions. As used herein, detection of programmable nuclease-based reporter cleavage (directly or indirectly) to determine the presence of a target nucleic acid sequence may be referred to as “DETECTR”.

[0228] The term, “detectable signal,” as used herein refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.

[0229] The term, “detecting a nucleic acid” and its grammatical equivalents, as usedDocket No.203477-779601 herein refers to detecting the presence or absence of the target nucleic acid in a sample that potentially contains the nucleic acid being detected.

[0230] The term, “effector protein,” as used herein refers to a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. In some embodiments, the complex comprises multiple effector proteins. In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some embodiments, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid. A non-limiting example of modifying a target nucleic acid is cleaving (hydrolysis) of a phosphodiester bond. Additional examples of modifying target nucleic acids are described herein and throughout. In some embodiments, the term, “effector protein” refers to a protein that is capable of modifying a nucleic acid molecule (e.g., by cleavage, deamination, recombination). Modifying the nucleic acid may modulate the expression of the nucleic acid molecule (e.g., increasing or decreasing the expression of a nucleic acid molecule). The effector protein may be a Cas protein (i.e., an effector protein of a CRISPR-Cas system).

[0231] The term, “guide nucleic acid,” as used herein refers to a nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of being non-covalently bound by an effector protein. The first sequence may be referred to herein as a spacer sequence. The second sequence may be referred to herein as a repeat sequence. In some embodiments, the first sequence is located 5’ of the second nucleotide sequence. In some embodiments, the first sequence is located 3’ of the second nucleotide sequence.

[0232] The terms, “non-naturally occurring” and “engineered,” as used herein are used interchangeably and indicate the involvement of human intervention. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and / or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non- limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, anDocket No.203477-779601 effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by human intervention.

[0233] The term, “protospacer adjacent motif (PAM),” as used herein refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. A PAM sequence may be required for a complex having an effector protein and a guide nucleic acid to hybridize to and modify the target nucleic acid. However, a given effector protein may not require a PAM sequence being present in a target nucleic acid for the effector protein to modify the target nucleic acid.

[0234] The terms, “reporter” and “reporter nucleic acid,” are used interchangeably herein to refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein.

[0235] The term, “sample,” as used herein generally refers to something comprising a target nucleic acid. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of non-limiting example, samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.

[0236] The term, “target nucleic acid,” as used herein refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single- stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded (e.g., double- stranded DNA).

[0237] The term, “target sequence,” as used herein when used in reference to a target nucleic acid refers to a sequence of nucleotides that hybridizes to a portion (preferably an equal length portion) of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.

[0238] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Docket No.203477-779601 ILLUSTRATIVE EMBODIMENTS

[0239] The present disclosure provides the following illustrative embodiments.

[0240] Embodiment 1. A system for measuring an amount of a target nucleic acid in a sample, the system comprising a plurality of reaction mixtures, wherein: (a) each reaction mixture of the plurality of reaction mixtures comprises a programmable nuclease, a guide nucleic acid, and a reporter; (b) the guide nucleic acid comprises a sequence configured to bind to the target nucleic acid; (c) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (d) cleavage of the reporter is effective to produce a detectable product at a rate proportional to the amount of the target nucleic acid present in the sample; (e) the plurality of reaction mixtures comprises control reaction mixtures comprising a predetermined amount of the target nucleic acid; (f) the predetermined amount of target nucleic acid in a first of the control reaction mixtures is at least 10-fold higher than the predetermined amount of target nucleic acid in a second of the control reaction mixtures; and (g) the plurality of reaction mixtures comprises a sample reaction mixture comprising a nucleic acid sample to be measured.

[0241] Embodiment 2. The system of Embodiment 1, wherein the control reaction mixtures comprise three or more control reaction mixtures, wherein each of the three or more control reaction mixtures comprises a different predetermined amount of the target nucleic acid.

[0242] Embodiment 3. The system of Embodiment 1 or 2, wherein the predetermined amount of target nucleic acid in the first of the control reaction mixtures is 10,000-fold higher than the predetermined amount of target nucleic acid in the second of the control reaction mixtures.

[0243] Embodiment 4. The system of any one of Embodiments 1-3, wherein each reaction mixture of the plurality of reaction mixtures further comprises reagents for amplifying the target nucleic acid.

[0244] Embodiment 5. The system of any one of Embodiments 1-4, wherein each reaction mixture of the plurality of reaction mixtures further comprises reagents for reverse transcribing the target nucleic acid.Docket No.203477-779601

[0245] Embodiment 6. The system of any one of Embodiments 1-5, wherein the plurality of reaction mixtures is contained in a microfluidic device.

[0246] Embodiment 7. The system of Embodiment 6, wherein the microfluidic device comprises a sample interface configured to receive a sample, and reaction chambers in fluid communication with the sample interface, wherein each reaction chamber comprises one of the reaction mixtures of the plurality of reaction mixtures.

[0247] Embodiment 8. The system of Embodiment 7, wherein the microfluidic device comprises an input chamber fluidically connected between the sample interface and the reaction chambers, optionally wherein the sample interface, input chamber, and reaction chambers are fluidically connected by one or more valves.

[0248] Embodiment 9. The system of Embodiment 8, wherein the input chamber comprises lysis reagents.

[0249] Embodiment 10. The system of any one of Embodiments 1-9, further comprising one or more lateral flow assay strips.

[0250] Embodiment 11. The system of any one of Embodiments 1-10, wherein the programmable nuclease is a Cas protein, optionally wherein the Cas protein is a Cas12 protein, a Cas13 protein, a Cas14 protein, or a CasPhi protein.

[0251] Embodiment 12. The system of any one of Embodiments 1-11, wherein the reporter is coupled to the guide nucleic acid.

[0252] Embodiment 13. The system of any one of Embodiments 1-12, wherein the reporter comprises a fluorescent label and a quencher.

[0253] Embodiment 14. The system of any one of Embodiments 1-13, wherein each reaction mixture of the plurality of reaction mixtures comprises one or more of glycerol, sucrose, or a tRNA.

[0254] Embodiment 15. A method for measuring an amount of a target nucleic acid in a sample with the system of any one of Embodiments 1-14, the method comprising: (a) initiating detection reactions by heating the plurality of reaction mixtures to a target temperature; (b) measuring the detectable product at each of a plurality of times for each of the plurality of reaction mixtures; (c) determining a time to result (TTR) for each of the plurality of reaction mixtures, wherein the TTR is a reaction time to reach a predetermined state; (d) comparing the TTR for the sample reaction mixture to the TTRs of the control reaction mixtures; andDocket No.203477-779601 (e) determining an amount of the target nucleic acid in the sample reaction mixture based on the comparing.

[0255] Embodiment 16. The method of Embodiment 15, wherein the TTR is a time from the initiating the detection reactions to reach a predetermined rate of increase in formation of the detectable product.

[0256] Embodiment 17. The method of Embodiment 15, wherein the TTR is a time from the initiating the detection reactions to reach a predetermined percent of a maximum level of detectable product formed at a last of the plurality of times.

[0257] Embodiment 18. The method of Embodiment 17, wherein the predetermined percent is at least 20%, 25%, or 30% of the maximum level.

[0258] Embodiment 19. The method of any one of Embodiments 15-18, further comprising using the TTR to generate a standard curve for measuring the target nucleic acid.

[0259] Embodiment 20. The method of Embodiment 19, wherein the standard curve is about linear on a log scale over a range of amounts of the target nucleic acid per reaction mixture, wherein the range of amounts span at least 2 logs, 3 logs, 4 logs, 5 logs, 6 logs, 7 logs, or 8 logs.

[0260] Embodiment 21. The method of Embodiment 19, wherein the standard curve is about linear on a log scale over a range of amounts of the target nucleic acid per reaction mixture, wherein the range of amounts comprises 1000, 100, 50, 25, or fewer copies of the target nucleic acid.

[0261] Embodiment 22. The method of Embodiment 20 or 21, wherein the standard curve that is about linear on a log scale has an R2 value of at least 0.85, 0.9, or 0.95 over the range of amounts.

[0262] Embodiment 23. The method of any one of Embodiments 20-22, wherein the TTRs for an amount within the range of amounts is about 1-60 minutes, 1-30 minutes, 1-20 minutes, 1-10 minutes, or 1-5 minutes.

[0263] Embodiment 24. The method of any one of Embodiments 15-23, wherein the detection reactions comprise amplification reactions to amplify the target nucleic acid.

[0264] Embodiment 25. The method of Embodiment 24, wherein the amplification reactions comprise loop mediated amplification (LAMP).

[0265] Embodiment 26. The method of any one of Embodiments 15-25, wherein the detection reactions comprise reverse transcription reactions.

[0266] Embodiment 27. The method of any one of Embodiments 15-26, wherein the detection reactions comprise cleaving the reporter in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid.Docket No.203477-779601

[0267] Embodiment 28. The method of any one of Embodiments 15-27, wherein: (i) the reporter comprises a fluorescent label and a quencher; and (ii) measuring the detectable product comprises measuring a level of fluorescence in the reaction mixtures. EXAMPLES

[0268] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art. Example 1: Relative Quantitation Standard Curve Generation using OnePot DETECTR Assay

[0269] Time to result (TTR) values were calculated using RT-LAMP-DETECTR one-pot assays to generate a relative quantitation standard curve for RSV-B. Briefly, various starting concentrations of purified RSV-B viral RNA target substrate was added to a reaction mixture comprising RT-LAMP-DETECTR trans cleavage buffer, CasM.21526 effector proteins (SEQ ID NO: 34), guide RNAs (SEQ ID NO: 73), RSV LAMP primer mix (SEQ ID NOs: 74-79), dNTPs, SYTO9, RNase Inhibitor, Bst 2.0 DNA polymerase, glycerol-free Warmstart RTx reverse transcriptase, TIPP, and an FQ reporter. The 1x RT-LAMP-DETECTR trans-cleavage buffer comprised: 20mMTris HCl pH8.8, 50mM KoAc, 10mM NH4SO4, 0.1% Tween 20, 5mM MgOAc. The FQ reporter used was an Alexa 594-labeled reporter (SEQ ID NO: 80). Reactions were carried out at 58ºC for 60 minutes. RT-LAMP activity was monitored by SYTO9 fluorescence signal generated during amplification and trans-cleavage activity was monitored by Alexa594 fluorescence signal upon cleavage of a fluorophore-quencher reporter by the activated RNP.

[0270] FIGS.2A-2B show RT-LAMP product generation (top) and detection of the amplified target nucleic acids by the effector proteins (bottom) in the one-pot RT-LAMP trans- cleavage reaction for a variety of different starting concentrations of RSV-B viral RNA. Time to result (TTR) was calculated for each concentration using a “consecutive increase method”, whereby the TTR was defined as the time at which 4 consecutive increasing points of fluorescence on the DETECTR curve have a slope of at least 1.05. Dose-dependent assay times were observed and it was hypothesized that the TTR could be used to generate a standard curve for target RSV-B concentration quantitation of an unknown sample. Importantly, for each targetDocket No.203477-779601 concentration, the effector protein was able to detect the target and generate sufficient fluorescence signal so to determine dose-dependent TTRs within about 20 minutes or less, under the conditions tested, even when non-specific RT-LAMP product was generated (as in the 0 copies per reaction negative control conditions (top left)). FIG.3 shows a standard curve generated using the 100cp / rxn – 10,000cp / rxn TTR data from FIGS.2A-2B. The TTR values (y- axis) were plotted against log-transformed target RSV-B RNA copy numbers (x-axis). The standard curve generated had a linear fit R2value of 89.6% and a quadratic fit R2value of 91%. These data show the feasibility of quantification using a one-pot reaction with a 3-log range (102to 104) under the conditions tested.

[0271] A similar experiment was run using a CasM.1895868 effector protein (SEQ ID NO: 81) and an Altair-00020 effector protein (SEQ ID NO: 72), and a saturation effect was observed at higher copy numbers, suggesting that slowing down the reaction may improve resolution.

[0272] TABLE 4 – Exemplary Reagents for RT-LAMP-DETECTR one-pot assaysDocket No.203477-779601Docket No.203477-779601Docket No.203477-779601Example 2: Effects of Glycerol Concentration on Relative Quantitation Standard Curve Generation using OnePot DETECTR Assay

[0273] Additives such as glycerol can be used to reduce the speed of a one-pot reaction. As discussed in Example 1, when the effector protein was changed from SEQ ID NO: 34 to SEQ ID NO: 81, a saturation effect was observed at higher copy numbers, suggesting that slowing the reaction down may improve resolution. Glycerol was introduced into the reaction mixture at 0% (control), 1%, and 5% v / v and the RT-LAMP-DETECTR one-pot assay was run as described in Example 1 with CasM.1895868 effector proteins (SEQ ID NO: 81) at 53ºC with various starting concentrations of RSV-B target RNA (0 copies (0cp / rxn), 50 copies (50cp / rxn), 100 copies (100cp / rxn), 500 copies (500cp / rxn), 1000 copies (1000cp / rxn), 5000 copies (5000cp / rxn), 10000 copies (10000cp / rxn), or 100000 copies (100000cp / rxn)).

[0274] Briefly, various starting concentrations of purified RSV-B viral RNA target substrate was added to a reaction mixture comprising RT-LAMP-DETECTR trans cleavage buffer, CasM.1895868 effector proteins (SEQ ID NO: 81), guide RNAs (SEQ ID NO: 73), RSV LAMP primer mix (SEQ ID NOs: 74-79), dNTPs, SYTO9, RNase Inhibitor, Bst 2.0 DNA polymerase, glycerol-free Warmstart RTx reverse transcriptase, TIPP, glycerol (0%, 1%, or 5%) and an FQ reporter. The 1x RT-LAMP-DETECTR trans-cleavage buffer comprised: 20mMTris HCl pH8.8, 50mM KoAc, 10mM NH4SO4, 0.1% Tween 20, 5mM MgOAc. The FQ reporter used was an Alexa 594-labeled reporter: 5Alex594N / TTATTATT / 3IAbRQSp / (SEQ ID NO: 80). Reactions were carried out at 53ºC for 60 minutes. RT-LAMP activity was monitored by SYTO9 fluorescence signal generated during amplification and trans-cleavage activity was monitored by Alexa594 fluorescence signal upon cleavage of a fluorophore-quencher reporter by the activated RNP. TTR was calculated for each concentration using a “threshold method”, whereby the TTR was defined as the time at which the DETECTR fluorescence reached 30% of its max fluorescence signal. FIG.4 shows standard curves generated for using the 50cp / rxn – 100,000cp / rxn TTR data calculated for each concentration of glycerol. The TTR values (y-axis) were plotted against log-transformed target RSV-B RNA copy numbers (x-axis). The standard curve generated for 0% glycerol had a linear fit R2value of 84% while 1% glycerol and 5%Docket No.203477-779601 glycerol improved the linear fit to R2values of 95% and 96%, respectively, indicating that glycerol improved the quantification range of the assay to a 4-log range (102to 105) under the conditions tested. These data show that, at least for some effector proteins and / or reagent sets, the addition of glycerol can slow the TTR of the one-pot reaction and improve resolution of the assay along a greater quantification range. Example 3: Effects of Sucrose Concentration on Relative Quantitation Standard Curve Generation using OnePot DETECTR Assay

[0275] Additives such as sucrose can be used to reduce the speed of a one-pot reaction. As discussed in Example 1, when the effector protein was changed from SEQ ID NO: 34 to SEQ ID NO: 81, a saturation effect was observed at higher copy numbers, suggesting that slowing the reaction down may improve resolution. Sucrose was introduced into the reaction mixture at 0% (control), 1%, 5%, and 10% v / v and the RT-LAMP-DETECTR one-pot assay was run as described in Example 1 with CasM.1895868 effector proteins (SEQ ID NO: 81) at 53ºC with various starting concentrations of RSV-B target RNA (0 copies (0cp / rxn), 50 copies (50cp / rxn), 100 copies (100cp / rxn), 500 copies (500cp / rxn), 1000 copies (1000cp / rxn), 5000 copies (5000cp / rxn), 10000 copies (10000cp / rxn), or 100000 copies (100000cp / rxn)).

[0276] Briefly, various starting concentrations of purified RSV-B viral RNA target substrate was added to a reaction mixture comprising RT-LAMP-DETECTR trans cleavage buffer, CasM.1895868 effector proteins (SEQ ID NO: 81), guide RNAs (SEQ ID NO: 73), RSV LAMP primer mix (SEQ ID NOs: 74-79), dNTPs, SYTO9, RNase Inhibitor, Bst 2.0 DNA polymerase, glycerol-free Warmstart RTx reverse transcriptase, TIPP, sucrose (0%, 1%, 5%, or 10%) and an FQ reporter. The 1x RT-LAMP-DETECTR trans-cleavage buffer comprised: 20mMTris HCl pH8.8, 50mM KoAc, 10mM NH4SO4, 0.1% Tween 20, 5mM MgOAc. The FQ reporter used was an Alexa 594-labeled reporter: 5Alex594N / TTATTATT / 3IAbRQSp / (SEQ ID NO: 80). Reactions were carried out at 53ºC for 60 minutes. RT-LAMP activity was monitored by SYTO9 fluorescence signal generated during amplification and trans-cleavage activity was monitored by Alexa594 fluorescence signal upon cleavage of a fluorophore-quencher reporter by the activated RNP. TTR was calculated for each concentration using a “threshold method”, whereby the TTR was defined as the time at which the DETECTR fluorescence reached 30% of its max fluorescence signal. FIG.5 shows standard curves generated for using the 50cp / rxn – 100,000cp / rxn TTR data calculated for each concentration of sucrose. The TTR values (y-axis) were plotted against log-transformed target RSV-B RNA copy numbers (x-axis). The standard curve generated for 0% sucrose had a linear fit R2value of 92% while 1% sucrose, 5% sucrose, and 10% sucrose improved the linear fit to R2values of 94%, 94%, and 95%, respectively,Docket No.203477-779601 indicating that sucrose improved the quantification range of the assay to a 4-log range (102to 105) under the conditions tested. These data show that, at least for some effector proteins and / or reagent sets, the addition of sucrose can slow the TTR of the one-pot reaction and improve resolution of the assay along a greater quantification range. Example 4: Effects of Polymerase Concentration on Relative Quantitation Standard Curve Generation using OnePot DETECTR Assay

[0277] Reducing the concentration of polymerase, in this case Bst 2.0 DNA polymerase, can be used to reduce the speed of a one-pot reaction. As discussed in Example 1, when the effector protein was changed from SEQ ID NO: 34 to SEQ ID NO: 81, a saturation effect was observed at higher copy numbers, suggesting that slowing the reaction down may improve resolution. The concentration of Bst 2.0 DNA polymerase was reduced from 6.0 Units (as in Example 1) to 1.0 Unit (6.0, 5.0, 4.0, 3.0, 2.0, 1.75, 1.5, 1.25, and 1.0 Units) and the RT-LAMP- DETECTR one-pot assay was run as described in Example 1 with CasM.1895868 effector proteins (SEQ ID NO: 81) at 58ºC with various starting concentrations of RSV-B target RNA (0 copies (0cp / rxn), 100 copies (100cp / rxn, 2-log concentration), 1000 copies (1000cp / rxn, 3-log concentration), 10000 copies (10000cp / rxn, 4-log concentration), 100000 copies (100000cp / rxn, 5-log concentration), or 1000000 copies (1000000cp / rxn, 6-log concentration).

[0278] Briefly, various starting concentrations of purified RSV-B viral RNA target substrate was added to a reaction mixture comprising RT-LAMP-DETECTR trans cleavage buffer, CasM.1895868 effector proteins of (SEQ ID NO: 81), guide RNAs (SEQ ID NO: 73), RSV LAMP primer mix (SEQ ID NOs: 74-79), dNTPs, SYTO9, RNase Inhibitor, varying amounts of Bst 2.0 DNA polymerase (6.0, 5.0, 4.0, 3.0, 2.0, 1.75, 1.5, 1.25, and 1.0 Units), glycerol-free Warmstart RTx reverse transcriptase, TIPP, and an FQ reporter. The 1x RT- LAMP-DETECTR trans-cleavage buffer comprised: 20mMTris HCl pH8.8, 50mM KoAc, 10mM NH4SO4, 0.1% Tween 20, 5mM MgOAc. The FQ reporter used was an Alexa 594- labeled reporter: 5Alex594N / TTATTATT / 3IAbRQSp / (SEQ ID NO: 80). Reactions were carried out at 58ºC for 60 minutes. RT-LAMP activity was monitored by SYTO9 fluorescence signal generated during amplification and trans-cleavage activity was monitored by Alexa594 fluorescence signal upon cleavage of a fluorophore-quencher reporter by the activated RNP. TTR was calculated for each concentration using a “consecutive increase method”, whereby the TTR was defined as the time at which 4 consecutive increasing points of fluorescence on the DETECTR curve have a slope of at least 1.05. It was initially observed that reducing the Bst 2.0 DNA polymerase amount from 6.0U to 2.0U or 1.0U resulted in improved standard curve linearity under the conditions tested (data not shown), so further tests were performed toDocket No.203477-779601 determine if there was an optimal concentration of Bst 2.0 DNA polymerase within this range. FIG.14 shows standard curves generated for using the 100cp / rxn – 1,000,000cp / rxn TTR data calculated for 1.75, 1.5, 1.25, and 1.0 Units of Bst 2.0 DNA polymerase. The TTR values (y- axis) were plotted against log-transformed target RSV-B RNA copy numbers / dilution (x-axis). The standard curve generated for 1.75U Bst 2.0 DNA polymerase had an improved linear fit R2value of 95.5% while 1.5U Bst 2.0 DNA polymerase, 1.25U Bst 2.0 DNA polymerase, and 1.0U Bst 2.0 DNA polymerase had improved the linear fit to R2values of 94.1%, 93.7%, and 91.6%, respectively, indicating that reduced polymerase concentration improved the quantification range of the assay to a 5-log range (102to 106) under the conditions tested. These data show that, at least for some effector proteins and / or reagent sets, the amount of polymerase can be adjusted so as to slow the TTR of the one-pot reaction and improve resolution of the assay along a greater quantification range.

[0279] Similar experiments were run using the Altair-00020 effector protein (SEQ ID NO: 72) to quantify RSV-B or KRAS target RNA, and a similar improvement in assay resolution was observed. Example 5: Effects of dNTP and Mg2+ Concentration on Relative Quantitation Standard Curve Generation using OnePot DETECTR Assay

[0280] Increasing the concentration of Mg2+ and dNTPs in the reaction mix can be used to reduce the speed of a one-pot reaction. As discussed in Example 1, when the effector protein was changed from SEQ ID NO: 34 to SEQ ID NO: 72, a saturation effect was observed at higher copy numbers, suggesting that slowing the reaction down may improve resolution. The concentration of Mg2+ was increased from 5 mM MgOAc (as in Example 1) to 8 mM MgOAc (5mM, 6mM, 7mM, 8mM), the concentration of dNTPs was increased from 1mM (as in Example 1) to 1.6 mM (1mM, 1.2mM, 1.4mM, 1.6mM), the concentration of Bst 2.0 polymerase was increased to 1.5 U (as in Example 4), and the RT-LAMP-DETECTR one-pot assay was run as described in Example 4 with Altair-00020 effector proteins (SEQ ID NO: 72) at 58ºC with various starting concentrations of KRAS in vitro transcribed (IVT) target RNA (0 copies (0cp / rxn), 100 copies (100cp / rxn, 3-log concentration), 1000 copies (1000cp / rxn, 4-log concentration), 10000 copies (10000cp / rxn, 5-log concentration), 100000 copies (100000cp / rxn, 6-log concentration), 1000000 copies (1000000cp / rxn, 7-log concentration), 10000000 copies (10000000cp / rxn, 8-log concentration), or 100000000 copies (100000000cp / rxn, 9-log concentration).

[0281] Briefly, various starting concentrations of in vitro transcribed KRAS RNA target substrate was added to a reaction mixture comprising RT-LAMP-DETECTR trans cleavageDocket No.203477-779601 buffer, Altair-00020 effector proteins (SEQ ID NO: 72), guide RNAs (SEQ ID NO: 82), KRAS LAMP primer mix (SEQ ID NOs: 83-88), varying concentrations of dNTPs (1mM, 1.2mM, 1.4mM, 1.6mM), SYTO9, RNase Inhibitor, Bst 2.0 DNA polymerase (1.5 Units), glycerol-free Warmstart RTx reverse transcriptase, TIPP, and an FQ reporter. The 1x RT-LAMP-DETECTR trans-cleavage buffer comprised: 20mMTris HCl pH8.8, 50mM KoAc, 10mM NH4SO4, 0.1% Tween 20, and varying concentrations of MgOAc (5mM, 6mM, 7mM, 8mM). The FQ reporter used was an Alexa 594-labeled reporter: 5Alex594N / CCCCCCCCCCCC / 3IAbRQSp / (SEQ ID NO: 89). Reactions were carried out at 58ºC for 60 minutes. RT-LAMP activity was monitored by SYTO9 fluorescence signal generated during amplification and trans-cleavage activity was monitored by Alexa594 fluorescence signal upon cleavage of a fluorophore-quencher reporter by the activated RNP. TTR was calculated for each concentration using a “consecutive increase method”, whereby the TTR was defined as the time at which 4 consecutive increasing points of fluorescence on the DETECTR curve have a slope of at least 1.05. FIG.15 shows standard curves generated for using the 100cp / rxn – 100,000,000cp / rxn TTR data calculated for 5mM MgOAc / 1mM dNTP, 7mM MgOAc / 1.2mM dNTP, and 7mM MgOAc / 1.4mM dNTP. The TTR values (y-axis) were plotted against log-transformed target KRAS IVT RNA copy numbers / dilution (x-axis). The standard curve generated for 5mM MgOAc / 1mM dNTP had a slope of -1.41 and an improved linear fit R2value of 0.94. The standard curve generated for 7mM MgOAc / 1.2mM dNTP had a slope of -5.05 and an improved linear fit R2value of 0.97. The standard curve generated for 7mM MgOAc / 1.4mM dNTP had a slope of -2.65 and an improved linear fit R2value of 0.98. In addition to changing the speed of the reaction and improving dispersion of the DETECTR curves, it was observed that changing the concentration of Mg2+ and / or dNTPs improved the max signal achieved by the DETECTR reactions, which was associated with higher R2values. These results indicate that increased Mg2+ and / or dNTP concentration improved the quantification range of the assay to a 7-log range (102to 108) under the conditions tested. These data show that, at least for some effector proteins and / or reagent sets, the amount of Mg2+ and / or dNTPs can be adjusted so as to slow the TTR of the one-pot reaction and improve resolution of the assay along a greater quantification range. Example 6: Effects of Temperature Changes on Relative Quantitation Standard Curve Generation using OnePot DETECTR Assay

[0282] Quantitative methods like qPCR can be limited in their ability to tolerate changes in operating temperatures, often utilizing specialized and / or expensive equipment to ensure accurate results. A screen was carried out to determine how tolerant the OnePot DETECTR assay and quantitation method described in Examples 1-5 was to temperature changes. TheDocket No.203477-779601 concentration of Bst 2.0 polymerase ...

Claims

Docket No.203477-779601 CLAIMS WHAT IS CLAIMED IS:

1. A system for measuring an amount of a target nucleic acid in a sample, the system comprising a plurality of reaction mixtures, wherein: (a) each reaction mixture of the plurality of reaction mixtures comprises a programmable nuclease, a guide nucleic acid, and a reporter; (b) the guide nucleic acid comprises a sequence configured to bind to the target nucleic acid; (c) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (d) cleavage of the reporter is effective to produce a detectable product at a rate proportional to the amount of the target nucleic acid present in the sample; (e) the plurality of reaction mixtures comprises control reaction mixtures comprising a predetermined amount of the target nucleic acid; (f) the predetermined amount of target nucleic acid in a first of the control reaction mixtures is at least 10-fold higher than the predetermined amount of target nucleic acid in a second of the control reaction mixtures; and (g) the plurality of reaction mixtures comprises a sample reaction mixture comprising a nucleic acid sample to be measured.

2. The system of claim 1, wherein the control reaction mixtures comprise three or more control reaction mixtures, wherein each of the three or more control reaction mixtures comprises a different predetermined amount of the target nucleic acid.

3. The system of claim 1, wherein the predetermined amount of target nucleic acid in the first of the control reaction mixtures is 10,000-fold higher than the predetermined amount of target nucleic acid in the second of the control reaction mixtures.

4. The system of claim 1, wherein each reaction mixture of the plurality of reaction mixtures further comprises reagents for amplifying the target nucleic acid.

5. The system of claim 1, wherein each reaction mixture of the plurality of reaction mixtures further comprises reagents for reverse transcribing the target nucleic acid.Docket No.203477-779601 6. The system of claim 1, wherein the plurality of reaction mixtures is contained in a microfluidic device.

7. The system of claim 6, wherein the microfluidic device comprises a sample interface configured to receive a sample, and reaction chambers in fluid communication with the sample interface, wherein each reaction chamber comprises one of the reaction mixtures of the plurality of reaction mixtures.

8. The system of claim 7, wherein the microfluidic device comprises an input chamber fluidically connected between the sample interface and the reaction chambers, optionally wherein the sample interface, input chamber, and reaction chambers are fluidically connected by one or more valves.

9. The system of claim 8, wherein the input chamber comprises lysis reagents.

10. The system of claim 1, further comprising one or more lateral flow assay strips.

11. The system of claim 1, wherein the programmable nuclease is a Cas protein, optionally wherein the Cas protein is a Cas12 protein, a Cas13 protein, a Cas14 protein, or a CasPhi protein.

12. The system of claim 1, wherein the reporter is coupled to the guide nucleic acid.

13. The system of claim 1, wherein the reporter comprises a fluorescent label and a quencher.

14. The system of claim 1, wherein each reaction mixture of the plurality of reaction mixtures comprises one or more of glycerol, sucrose, or a tRNA.

15. A method for measuring an amount of a target nucleic acid in a sample with the system of any one of claims 1-14, the method comprising: (a) initiating detection reactions by heating the plurality of reaction mixtures to a target temperature; (b) measuring the detectable product at each of a plurality of times for each of the plurality of reaction mixtures; (c) determining a time to result (TTR) for each of the plurality of reaction mixtures, wherein the TTR is a reaction time to reach a predetermined state; (d) comparing the TTR for the sample reaction mixture to the TTRs of the control reaction mixtures; and (e) determining an amount of the target nucleic acid in the sample reaction mixture based on the comparing.Docket No.203477-779601 16. The method of claim 15, wherein the TTR is a time from the initiating the detection reactions to reach a predetermined rate of increase in formation of the detectable product.

17. The method of claim 15, wherein the TTR is a time from the initiating the detection reactions to reach a predetermined percent of a maximum level of detectable product formed at a last of the plurality of times.

18. The method of claim 17, wherein the predetermined percent is at least 20%, 25%, or 30% of the maximum level.

19. The method of claim 15, further comprising using the TTR to generate a standard curve for measuring the target nucleic acid.

20. The method of claim 19, wherein the standard curve is about linear on a log scale over a range of amounts of the target nucleic acid per reaction mixture, wherein the range of amounts span at least 2 logs, 3 logs, 4 logs, 5 logs, 6 logs, 7 logs, or 8 logs.

21. The method of claim 19, wherein the standard curve is about linear on a log scale over a range of amounts of the target nucleic acid per reaction mixture, wherein the range of amounts comprises 1000, 100, 50, 25, or fewer copies of the target nucleic acid.

22. The method of claim 20, wherein the standard curve that is about linear on a log scale has an R2value of at least 0.85, 0.9, or 0.95 over the range of amounts.

23. The method of claim 20, wherein the TTRs for an amount within the range of amounts is about 1-60 minutes, 1-30 minutes, 1-20 minutes, 1-10 minutes, or 1-5 minutes.

24. The method of claim 15, wherein the detection reactions comprise amplification reactions to amplify the target nucleic acid.

25. The method of claim 24, wherein the amplification reactions comprise loop mediated amplification (LAMP).

26. The method of claim 15, wherein the detection reactions comprise reverse transcription reactions.

27. The method of claim 15, wherein the detection reactions comprise cleaving the reporter in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid.Docket No.203477-779601 28. The method of claim 15, wherein: (i) the reporter comprises a fluorescent label and a quencher; and (ii) measuring the detectable product comprises measuring a level of fluorescence in the reaction mixtures.

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