SINGLE BUFFET COMPOSITIONS FOR THE DETECTION OF NUCLEAR ACIDS

Description

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 125,384, filed on December 14, 2020; U.S. Provisional Application No. 63 / 166,923, filed on March 26, 2021; U.S. Provisional Application No. 63 / 239,884 filed on September 1, 2021; U.S. Provisional Application No. 63 / 125,387 filed on December 14, 2020; U.S. Provisional Application No. 63 / 222,377 filed on July 15, 2021; U.S. Provisional Application No. 63 / 239,917 filed on September 1, 2021; and U.S. Provisional Application No. 63 / 151,592 filed on February 19, 2021.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Contract No. N66001-21-C-4048 awarded by the Department of Defense, Defense Advanced Research Projects Agency (DARPA). The US government has certain rights in the invention.BACKGROUND

[0003] 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. In particular, there is a need for rapid and low cost point-of-care testing for detection of viral infections, for example, including human immunodeficiency virus (HIV), SARS and MERS coronaviruses, influenza H1N1 virus, Ebola virus (EBOV), Zika virus (ZIKV), and SARS-CoV-2.

[0004] Despite decades of continuous and rapid development in nucleic acid diagnostics, nucleic acid detection is typically time intensive, error prone, and susceptible to contamination. Many of these problems stem from the multi-condition requirements requisite for multi-reaction detection schemes. Nucleic acid targets are often present in low copy numbers and as minor constituents in complex samples. Therefore, detection of these targets often requires amplification prior to detection.

[0005] However, amplification and detection reagents often comprise stringent condition requirements that are not cross-compatible, necessitating buffer exchange and sample transfer steps that can increase time and user input requirements and result in contamination and sample volume loss.

[0006] WO 2020 / 028729 A1 describes programmable nuclease compositions and methods of use thereof.

[0007] WO 2018 / 107129 A1 describes CRISPR effector system-based diagnostics.

[0008] WO 2021 / 252836 A1 describes CRISPR-based SARS-CoV-2 detection.

[0009] WO 2021 / 243308 A1 describes a programmable nuclease diagnostic device.SUMMARY

[0010] The invention is defined by appended claims 1, 14, 15, and 16. Various embodiments are defined in the appended dependent claims.

[0011] Described herein, in certain embodiments, is a system for detecting a target nucleic acid, comprising a buffer comprising: (i) reagents for an amplification reaction targeting the target nucleic acid; and (ii) reagents for a DETECTR reaction targeting the target nucleic acid, comprising a programmable nuclease, a non-naturally occurring guide nucleic acid, and a reporter, wherein the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid, wherein at least 1 nM of the reporter undergoes transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system. In some examples, at least 5 nM of the reporter undergoes transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system. In some examples, at least 10 nM of the reporter undergoes transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system. In some embodiments, at least 1 nM of the reporter undergoes transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, at least 5 nM of the reporter undergoes transcollateral cleavage within one hour of addition of at least 100 copies of the target nucleic acid to the system. In some embodiments, the time to completion for the amplification and DETECTR reactions is each less than 45 minutes when performed in the buffer. In some embodiments, the amplification and DETECTR reagents have half-lives of greater than 1 week at room temperature. In some embodiments, the activities of the amplification reagents and the DETECTR reagents diminishes by less than 10% following a freeze-thaw cycle. In some embodiments, the reagents for the amplification reaction comprise reagents for thermal cycling amplification. In some embodiments, the reagents for the amplification reaction comprise reagents for isothermal amplification. In some embodiments, the reagents for the amplification reaction comprise reagents for transcription mediated amplification (TMA), helicase dependent amplification (HDA), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), 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). In some embodiments, the reagents for the amplification reaction comprise reagents for loop mediated amplification (LAMP). In some embodiments, the system further comprises an activator for the amplification reaction. In some embodiments, the activator for the amplification reaction comprises a magnesium or calcium salt. In some embodiments, the programmable nuclease comprises at least 60% sequence identity to SEQ ID NO: 18-170 or 221-268. In some embodiments, the programmable nuclease comprises a RuvC catalytic domain. In some embodiments, the programmable nuclease is a type V CRISPR / Cas effector protein. In some embodiments, the type V CRISPR / Cas effector protein is a Cas12 protein. In some embodiments, the Cas12 protein comprises a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide. In some embodiments, the Cas12 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 18 - SEQ ID NO: 60. In some embodiments, the Cas12 protein is selected from SEQ ID NO: 18 - SEQ ID NO: 60. In some embodiments, the type V CRISPR / Cas effector protein is a Cas14 protein. In some embodiments, the Cas14 protein comprises a Cas14a polypeptide, a Cas14b polypeptide, a Cas14c polypeptide, a Cas14d polypeptide, a Cas14e polypeptide, a Cas14f polypeptide, a Cas14g polypeptide, a Cas14h polypeptide, a Cas14i polypeptide, a Cas14j polypeptide, or a Cas14k polypeptide. In some embodiments, the Cas14 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 61 - SEQ ID NO: 152. In some embodiments, the Cas14 protein is selected from SEQ ID NO: 61 - SEQ ID NO: 152. In some embodiments, the type V CRISPR / Cas effector protein is a CasΦ protein. In some embodiments, the CasΦ protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 221 - SEQ ID NO: 268. In some embodiments, the CasΦ protein is selected from SEQ ID NO: 221 - SEQ ID NO: 268. In some embodiments, the system further comprises a reverse transcriptase, an oligonucleotide primer, and dNTPs for reverse transcribing the target nucleic acid. In some embodiments, the buffer is a lysis buffer. In some embodiments, the viscosity of the buffer is at least 5 centipoise (cP). In some embodiments, the buffer comprises a pH of 7.5 to 8.5, at least 10 mM of a buffering agent, at least 1 mM ammonium acetate, at least 10 mM potassium acetate, at least 2.5 mM magnesium acetate, and at least 0.5% glycerol. In some embodiments, the buffer comprises a pH of 7.7 to 8.3. In some embodiments, the buffer comprises a pH of 7.85 to 8.15. In some embodiments, the buffering agent comprises HEPES, imidazole, TRIS-HCl, or phosphate. In some embodiments, the buffer further comprises at least 0.05% by volume of a detergent. In some embodiments, the detergent comprises Tween 20. In some embodiments, the buffer comprises a pH of 7.5 to 8.5, at least 5 mM of a buffering agent, at least 20 mM potassium acetate, at least 2.5 mM magnesium acetate, and at least 0.5% glycerol. In some embodiments, the buffer comprises a pH of 7.7 to 8.3. In some embodiments, the buffer comprises a pH of 7.85 to 8.15. In some embodiments, the buffering agent comprises phosphate or TRIS-HCl. In some embodiments, the buffer comprises at least 1 mM ammonium sulfate. In some embodiments, the buffer comprises at least 0.05% by volume of a detergent. In some embodiments, the detergent comprises Tween 20. In some embodiments, the buffer comprises a pH of 7.25 to 8.75, at least 5 mM of a buffering agent, at least 7.5 mM potassium acetate, at least 1 mM magnesium acetate, and at least 0.5% glycerol. In some embodiments, the buffering agent comprises phosphate. In some embodiments, the buffer comprises a pH of 7.5 to 8.5. In some embodiments, the buffer comprises a pH of 7.75 to 8.25. In some embodiments, the buffer further comprises at least 1 mM ammonium sulfate. In some embodiments, the buffer further comprises at least 0.05% by volume of a detergent. In some embodiments, the detergent comprises Tween 20.

[0012] The invention provides a system for detecting a target nucleic acid, comprising a buffer comprising: (i) amplification reagents for an amplification reaction targeting the target nucleic acid; and (ii) detection reagents for a detection reaction targeting the target nucleic acid; wherein the amplification reagents comprise one or more oligonucleotide primers, and a DNA polymerase; wherein the detection reagents comprise a programmable nuclease, a non-naturally occurring guide nucleic acid, and reporters; wherein the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid or DNA amplicons thereof; wherein the amplification reagents are present in amounts effective to amplify the target nucleic acid in a test sample to produce DNA amplicons of the target nucleic acid; wherein the programmable nuclease and non-naturally occurring guide nucleic acid form a complex in the buffer that is activated upon binding one of the DNA amplicons to induce detectable transcollateral cleavage of the reporters; and wherein the buffer is a lysis buffer. In some embodiments, (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system. In some embodiments, the amplification reagents and detection reagents are present in amounts effective to produce a detectable signal in less than 45 minutes in the presence of the target nucleic acid. In some embodiments, the amplification and detection reagents have half-lives of greater than 1 week at room temperature. In some embodiments, the activities of the amplification reagents and the detection reagents diminishes by less than 10% following a freeze-thaw cycle. In some embodiments, the amplification reagents comprise reagents for isothermal amplification. In some embodiments, the amplification reagents comprise reagents for transcription mediated amplification (TMA), helicase dependent amplification (HDA), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), 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). In some embodiments, the amplification reagents comprise reagents for loop mediated amplification (LAMP), and further wherein (a) the one or more primers comprise a first primer and a second primer targeted to the target nucleic acid; (b) the first primer comprises a 5' region that is complementary to a sequence generated by extension of the first primer; (c) the second primer comprises a 5' region that is complementary to a sequence generated by extension of the second primer; and (d) the DNA polymerase is a strand-displacing DNA polymerase. In some embodiments, the system further comprises an activator for the amplification reaction, such as a magnesium or calcium salt. In some embodiments, the programmable nuclease comprises at least 60% sequence identity to SEQ ID NO: 18-170 or 221-268. In some embodiments, the programmable nuclease comprises an RuvC catalytic domain. In some embodiments, the programmable nuclease is a type V CRISPR / Cas effector protein. In some embodiments, the type V CRISPR / Cas effector protein is a Cas12 protein, such as (a) a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide; (b) a protein that has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 18 - SEQ ID NO: 60; or (c) a protein having a sequence selected from SEQ ID NO: 18 - SEQ ID NO: 60. In some embodiments, the type V CRISPR / Cas effector protein is a Cas14 protein, such as (a) a Cas14a polypeptide, a Cas14b polypeptide, a Cas14c polypeptide, a Cas14d polypeptide, a Cas14e polypeptide, a Cas 14f polypeptide, a Cas14g polypeptide, a Cas14h polypeptide, a Cas14i polypeptide, a Cas14j polypeptide, or a Cas14k polypeptide; (b) a protein that has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 61 - SEQ ID NO: 152; or (c) a protein having a sequence selected from SEQ ID NO: 61 - SEQ ID NO: 152. In some embodiments, the type V CRISPR / Cas effector protein is a CasΦ protein, such as (a) a protein that has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 221 - SEQ ID NO: 268; or (b) a protein having a sequence selected from SEQ ID NO: 221 - SEQ ID NO: 268. In some embodiments, the system further comprises a reverse transcriptase, an oligonucleotide primer, and dNTPs for reverse transcribing the target nucleic acid. The buffer is a lysis buffer. In some embodiments, the viscosity of the buffer is at least 5 centipoise (cP). In some embodiments, the buffer comprises a pH of 7.5 to 8.5, at least 10 mM of a buffering agent, at least 1 mM ammonium acetate, at least 10 mM potassium acetate, at least 2.5 mM magnesium acetate, and at least 0.5% glycerol; and optionally: (a) the buffer comprises a pH of 7.7 to 8.3, or a pH of 7.85 to 8.15; (b) the buffering agent comprises HEPES, imidazole, TRIS-HCl, or phosphate; and / or (c) the buffer further comprises at least 0.05% by volume of a detergent, optionally wherein the detergent comprises Tween 20. In some embodiments, the buffer comprises a pH of 7.5 to 8.5, at least 5 mM of a buffering agent, at least 20 mM potassium acetate, at least 2.5 mM magnesium acetate, and at least 0.5% glycerol; and optionally: (a) the buffer comprises a pH of 7.7 to 8.3, or a pH of 7.85 to 8.15; (b) the buffering agent comprises phosphate or TRIS-HCl; (c) the buffer comprises at least 1 mM ammonium sulfate; and / or (d) the buffer comprises at least 0.05% by volume of a detergent, optionally wherein the detergent comprises Tween 20. In some embodiments, the buffer comprises a pH of 7.25 to 8.75, at least 5 mM of a buffering agent, at least 7.5 mM potassium acetate, at least 1 mM magnesium acetate, and at least 0.5% glycerol; and optionally: (a) the buffering agent comprises phosphate; (b) the buffer comprises a pH of 7.5 to 8.5, or a pH of 7.75 to 8.25; (c) the buffer further comprises at least 1 mM ammonium sulfate; and / or (d) the buffer further comprises at least 0.05% by volume of a detergent, optionally wherein the detergent comprises Tween 20.

[0013] In some embodiments, the system further comprises a circular template with internal complementarity formed from a single polynucleotide strand, wherein: (a) the circular template comprises a first portion with complementarity to one of the one or more oligonucleotide primers and a second portion with complementarity to a portion of the target nucleic acid; (b) the internal complementarity comprises part of the first portion and part of the second portion; (c) the second portion has a total length that is longer than a combined length of the first portion and second portion that are within the internal complementarity; and (d) the circular template undergoes a conformational change upon hybridization to the target nucleic acid to expose the first portion to hybridization to the oligonucleotide primer. In some embodiments, the system further comprises a circular template, wherein: (a) the circular template comprises a first portion with complementarity to one of the one or more oligonucleotide primers and a second portion with complementarity to the target nucleic acid; (b) the oligonucleotide primer complementary to the first portion comprises a blocking motif at its 3' end; and (c) the oligonucleotide primer complementary to the first portion undergoes cleavage to remove the blocking motif by the programmable nuclease in the presence of the target nucleic acid.

[0014] In some embodiments, the system further comprises a polymer matrix, wherein the polymer matrix is complexed with the reporters. In some embodiments, the polymer matrix is formed from copolymerization of at least a first plurality of monomers with the reporters. In some embodiments, the polymer matrix comprises a hydrogel.

[0015] In some embodiments, the buffer of system further comprises: (a) one or more of betaine monohydrate, acetamide, GABA, L-proline, beta-alanine, 6-aminohexanoic acid, urea, methylurea, ethylurea, hypotaurine, NDSB-256, and ammonium acetate; (b) one or more of trehalose, xylitol, D-sorbitol, sucrose, and trimethylamine N-oxide dihydrate; and / or (c) trimethylamine N-oxide dihydrate.

[0016] The invention provides a system for detecting a target nucleic acid, comprising reagents in a buffer, wherein (a) the reagents comprise hairpin polynucleotides, programmable nucleases, non-naturally occurring guide nucleic acids, and reporters; (b) each hairpin polynucleotide comprises one or more RNA loops, a first portion comprising DNA, and a second portion joined to the first portion by one of the one or more RNA loops; (c) each non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid; (d) in each hairpin polynucleotide, the second portion of the hairpin polynucleotide hybridizes to a segment of the first portion; (e) the programmable nucleases and non-naturally occurring guide nucleic acids form complexes in the buffer that are activated upon binding the target nucleic acid; (f) an activated programmable nuclease is effective to induce (i) transcollateral cleavage of the one or more RNA loops, and (ii) detectable transcollateral cleavage of the reporters; and (g) cleavage of the one or more RNA loops of one of the hairpin polynucleotides is effective to release the first portion of the hairpin polynucleotide to hybridize with one of the non-naturally occurring guide nucleic acids and form a further activated programmable nuclease. In some embodiments, the one or more RNA loops comprise a first RNA loop joining the first portion and the second portion, and a second RNA loop joining the first portion and a third portion; wherein the third portion hybridizes to a different segment of the first portion. In some embodiments, the second portion comprises RNA, DNA, or both.

[0017] In some embodiments described herein is a method of assaying for a target nucleic acid in a sample, the method comprising: (a) amplifying a portion of the target nucleic acid; (b) performing a DETECTR reaction targeting the target nucleic acid, comprising contacting the target nucleic acid with a programmable nuclease, a non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, and a reporter, wherein the contacting the sample to reagents for amplifying and the contacting the sample to reagents for the DETECTR reaction are performed in the same reaction volume; and (c) assaying for a change in a signal, wherein the change in the signal is produced by cleavage of the reporter. In some embodiments, the amplifying and the DETECTR reaction occur simultaneously. In some embodiments, the assaying comprises measuring the rate of the change in the signal. In some embodiments, the change in the signal identifies a concentration of the target nucleic acid in the sample. In some embodiments, the amplifying comprises thermal cycling amplification. In some embodiments, the amplifying comprises isothermal amplification. In some embodiments, the amplifying comprises transcription mediated amplification (TMA), helicase dependent amplification (HDA), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), 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). In some embodiments, the amplifying comprises loop mediated amplification (LAMP). In some embodiments, the amplifying comprises providing a plurality of primers that target different portions of the target nucleic acid. In some embodiments, the reagents for amplification comprise a forward inner primer (FIP) primer, a backward inner primer (BIP) primer, a forward loop primer (LF) primer, and a backward loop primer (LB primer). In some embodiments, the method comprises reverse transcribing the target nucleic acid. In some embodiments, the reverse transcribing comprises contacting the sample to reagents for reverse transcription, and wherein the reagents for reverse transcription comprise a reverse transcriptase, an oligonucleotide primer, and dNTPs. In some embodiments, the programmable nuclease comprises a RuvC catalytic domain. In some embodiments, the programmable nuclease has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 397 - SEQ ID NO: 423; or is selected from SEQ ID NO: 397- SEQ ID NO: 423.

[0018] In some embodiments, the programmable nuclease is a type V CRISPR / Cas effector protein. In some embodiments, the type V CRISPR / Cas effector protein is a Cas12 protein. In some embodiments, the Cas12 protein comprises a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide. In some embodiments, the Cas12 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 18 - SEQ ID NO: 60. In some embodiments, the Cas12 protein is selected from SEQ ID NO: 18 - SEQ ID NO: 60. In some embodiments, the type V CRISPR / Cas effector protein is a Cas14 protein. In some embodiments, the Cas14 protein comprises a Cas14a polypeptide, a Cas14b polypeptide, a Cas14c polypeptide, a Cas14d polypeptide, a Cas14e polypeptide, a Cas14f polypeptide, a Cas14g polypeptide, a Cas14h polypeptide, a Cas14i polypeptide, a Cas14j polypeptide, or a Cas14k polypeptide. In some embodiments, the Cas14 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 61 - SEQ ID NO: 152. In some embodiments, the Cas14 protein is selected from SEQ ID NO: 61 - SEQ ID NO: 152. In some embodiments, the type V CRISPR / Cas effector protein is a CasΦ protein. In some embodiments, the CasΦ protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 221 - SEQ ID NO: 268. In some embodiments, the CasΦ protein is selected from SEQ ID NO: 221 - SEQ ID NO: 268. In some embodiments, the method further comprises lysing a cell or virus comprising the target nucleic acid. In some embodiments, the lysing is performed in the same reaction volume as the amplifying and the DETECTR reaction. In some embodiments, the change in the signal comprises a change in a calorimetric, potentiometric, amperometric, or piezo-electric signal. In some embodiments, the change in the signal comprises a change in a colorimetric signal (e.g., an increase in intensity, a decrease in intensity, a change in color, etc.). In some embodiments, the change in the signal comprises a fluorescence signal (e.g., an increase in intensity, a decrease in intensity, a change in phase or wavelength, etc.). In some embodiments, the assaying comprises detecting the change in a signal with a smartphone. In some embodiments, the reaction volume comprises a viscosity of at least 5 cP. In some embodiments, the reaction volume comprises a total dissolved solids concentration of at least 200 mM.

[0019] The invention also provides a method of assaying for a target nucleic acid in a sample, the method comprising: (a) amplifying a portion of the target nucleic acid with a DNA polymerase to produce DNA amplicons of the target nucleic acid; (b) forming a complex comprising one of the DNA amplicons, a programmable nuclease, and a non-naturally occurring guide nucleic acid that hybridizes to a segment of the DNA amplicon, thereby activating the programmable nuclease; (c) cleaving reporters with the activated programmable nuclease; and (d) detecting a change in a signal, wherein the change in the signal is produced by cleavage of the reporters; wherein the target nucleic acid and reagents for the amplifying and cleaving are present in the same reaction volume; and wherein the method further comprises lysing a cell or virus comprising the target nucleic acid and wherein the lysing is performed in the same reaction volume as the amplifying and the cleaving. In some embodiments, the amplifying and the cleaving occur simultaneously. In some embodiments, the method further comprises measuring the rate of the change in the signal. In some embodiments, the method further comprises measuring a concentration of the target nucleic acid in the sample based on the change in the signal. In some embodiments, the amplifying comprises isothermal amplification. In some embodiments, the amplifying comprises transcription mediated amplification (TMA), helicase dependent amplification (HDA), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), 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). In some embodiments, the amplifying comprises loop mediated amplification (LAMP), wherein the LAMP comprises amplification with a first primer and a second primer targeted to the target nucleic acid and a strand-displacing polymerase, wherein the first primer comprises a 5' region that is complementary to a sequence generated by extension of the first primer, and wherein the second primer comprises a 5' region that is complementary to a sequence generated by extension of the second primer. In some embodiments, the amplifying comprises providing a plurality of primers that target different portions of the target nucleic acid. In some embodiments, reagents for the amplification comprise a FIP primer, a BIP primer, a LF primer, and a LB primer. In some embodiments, the amplifying comprises reverse transcribing the target nucleic acid. In some embodiments, the programmable nuclease comprises a RuvC catalytic domain. In some embodiments, the programmable nuclease has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 397 - SEQ ID NO: 423; or is selected from SEQ ID NO: 397- SEQ ID NO: 423.

[0020] In some embodiments, the programmable nuclease is a type V CRISPR / Cas effector protein. In some embodiments, the type V CRISPR / Cas effector protein is a Cas12 protein, such as (a) a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide; (b) a protein that has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 18 - SEQ ID NO: 60; or (c) a protein having a sequence selected from SEQ ID NO: 18 - SEQ ID NO: 60. In some embodiments, the type V CRISPR / Cas effector protein is a Cas14 protein, such as (a) a Cas14a polypeptide, a Cas14b polypeptide, a Cas14c polypeptide, a Cas14d polypeptide, a Cas14e polypeptide, a Cas14f polypeptide, a Cas14g polypeptide, a Cas14h polypeptide, a Cas14i polypeptide, a Cas14j polypeptide, or a Cas14k polypeptide; (b) a protein that has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 61 - SEQ ID NO: 152; or (c) a protein having a sequence selected from SEQ ID NO: 61 - SEQ ID NO: 152. In some embodiments, the type V CRISPR / Cas effector protein is a CasΦ protein, such as (a) a protein that has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 221 - SEQ ID NO: 268; or (b) a protein having a sequence selected from SEQ ID NO: 221 - SEQ ID NO: 268. The method further comprises lysing a cell or virus comprising the target nucleic acid. The lysing is performed in the same reaction volume as the amplifying and the cleaving. In some embodiments, the change in the signal comprises a change in a calorimetric, potentiometric, amperometric, or piezo-electric signal. In some embodiments, the change in the signal comprises a change in a colorimetric signal. In some embodiments, the change in the signal comprises a fluorescence signal. In some embodiments, the assaying comprises detecting the change in a signal with a smartphone. In some embodiments, the reaction volume comprises a viscosity of at least 5 cP. In some embodiments, the reaction volume comprises a total dissolved solids concentration of at least 200 mM. In some embodiments, the system further comprises Thermostable inorganic pyrophosphatase (TIPP). In some embodiments, the method further comprises signal enhancement via hydrolysis of inorganic pyrophosphates, such as by enzymatic hydrolysis. In some embodiments, the amplification and the cleaving are carried out in the presence of TIPP, and wherein the signal is enhanced by the hydrolysis of inorganic pyrophosphates by TIPP, as compared to the same reaction carried out without TIPP. In some embodiments, the reaction volume further comprises Thermostable inorganic pyrophosphatase (TIPP).

[0021] In some embodiments, the amplifying comprises amplification of a circular template with an oligonucleotide primer, and further wherein: (a) the circular template comprises a single polynucleotide strand having internal complementarity; (b) the circular template comprises a first portion with complementarity to the primer and a second portion with complementarity to a portion of the target nucleic acid; (c) the internal complementarity comprises part of the first portion and part of the second portion; (d) the second portion has a total length that is longer than a combined length of the first portion and second portion that are within the internal complementarity; (e) the circular template undergoes a conformational change upon hybridization to the target nucleic acid to expose the first portion to hybridization to the oligonucleotide primer; and (f) extension of the oligonucleotide primer along the circular template produces the DNA amplicons. In some embodiments, the amplifying comprises amplification of a circular template with an oligonucleotide primer, and further wherein: (a) the circular template comprises a first portion with complementarity to the oligonucleotide primer and a second portion with complementarity to the target nucleic acid; (b) the oligonucleotide primer complementary to the first portion comprises a blocking motif at its 3' end; (c) the oligonucleotide primer undergoes cleavage to remove the blocking motif by the programmable nuclease in the presence of the target nucleic acid; and (d) extension of the oligonucleotide primer along the circular template produces the DNA amplicons. In some embodiments, the amplification is isothermal. In some embodiments, the DNA polymerase is a strand-displacing polymerase.

[0022] In some embodiments of the methods, the reporters are complexed with a polymer matrix. In some embodiments, the polymer matrix is formed from copolymerization of a plurality of monomers with the reporters. In some embodiments, the polymer matrix comprises a hydrogel. In some embodiments, (i) the step of cleaving the reporters releases detectable moieties from the polymer matrix, and (ii) the step of detecting comprises capturing and detecting the released detectable moieties at a capture region of a support medium.

[0023] In some embodiments, said same reaction volume comprises one or more additives comprising: (a) one or more of betaine monohydrate, acetamide, GABA, L-proline, beta-alanine, 6-aminohexanoic acid, urea, methylurea, ethylurea, hypotaurine, NDSB-256, and ammonium acetate; (b) one or more of trehalose, xylitol, D-sorbitol, sucrose, and trimethylamine N-oxide dihydrate; and / or (c) trimethylamine N-oxide dihydrate.

[0024] The invention also provides a method of assaying for a target nucleic acid in a sample, the method comprising the following steps in a single reaction volume: (a) forming a complex comprising the target nucleic acid, a first programmable nuclease, and a first non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, thereby activating the programmable nuclease; (b) cleaving a hairpin polynucleotide of a plurality of hairpin polynucleotides with the activated programmable nuclease, wherein each hairpin polynucleotide comprises (i) one or more RNA loops that are cleaved, (i) a first portion comprising DNA, and (iii) a second portion joined to the first portion by one of the one or more RNA loops, wherein the second portion is hybridized to a segment of the first portion; (c) forming a second complex comprising the first portion of the cleaved hairpin polynucleotide, a second programmable nuclease, and a second non-naturally occurring guide nucleic acid that hybridizes to the first portion of the cleaved hairpin, thereby activating the second programmable nuclease; (d) cleaving reporters with the activated first or second programmable nuclease; and (e) detecting a change in a signal, wherein the change in the signal is produced by cleavage of the reporters. In some embodiments, the first programmable nuclease and the second programmable nuclease are the same, such as any of the programmable nucleases disclosed herein. In some embodiments, the one or more RNA loops comprise a first RNA loop joining the first portion and the second portion, and a second RNA loop joining the first portion and a third portion; wherein the third portion hybridizes to a different segment of the first portion. In some embodiments, the second portion comprises RNA, DNA, or both.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] The novel features of the disclosure are set forth with particularity in the appended claims. 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 (also "figure" and "FIG." herein), of which: FIG. 1 shows the mean time to result for RT-LAMP reactions performed in a variety of buffers. FIG. 2 provides summary effect sizes for different buffer constituents on the mean time to result for RT-LAMP reactions. The values on the right of the table provides the statistical significance for each variable or set of variables on the time to result relative to a commercial buffer control. FIG. 3 provides a graphical representation of measured effect sizes for various buffer components on RT-LAMP reaction times. The final column shows the weighting function used to generate the bottom ('Desirability') row of the chart, which shows whether a variable correlates positively or negatively with RT-LAMP reaction time. FIG. 4 shows the mean reaction times for DETECTR reactions performed with the buffers listed in TABLE 2, SEQ ID NO: 28 targeting SARS-CoV-2 N-gene, and two separate dilutions of SARS-CoV N-gene LAMP amplicons ('Dil 1' & 'Dil 2'). FIG. 5 provides effect summary sizes for the buffer constituents in the DETECTR assay summarized in FIG. 4. FIG. 6 provides a graphical representations of measured effect sizes for various buffer components on the DETECTR reaction times shown in FIG. 4. FIG. 7A - 7C graphically depicts RT-LAMP, DETECTR reaction times, and total reaction times (RT-LAMP + DETECTR reaction times) performed in a variety of buffers. FIG. 8 shows the mean reaction times for a variety of RT-LAMP assays performed in different buffers over seven (Dil-1 through Dil-7) replicates. NTC denotes controls lacking the target nucleic acid. FIG. 9 displays combined RT-LAMP and DETECTR assay times for 4 different buffer formulations. FIG. 10 provides the rates of RT-LAMP reactions performed in 10 different buffers. FIG. 11 provides combined RT-LAMP and DETECTR assay times for 4 different buffer systems and 3 different initial target nucleic acid copy numbers. FIG. 12A - 12B panel A displays reaction times for 4 different buffer systems and 3 different initial copy numbers for the target nucleic acid. Panel A provides reaction times for RT-LAMP reactions, while panel B provides reaction times for DETECTR assays. FIG. 13A - 13B shows the effect size and statistical significance of different buffer parameters on the rates of RT-LAMP reactions. The far right column shows the weighting function used to generate the bottom ('Desirability') row of the chart, which depicts whether a variable correlates positively or negatively with RT-LAMP reaction time. FIG. 14A - 14B shows the effect size and statistical significance of buffer parameters on the rates of DETECTR reactions. The far right column shows the weighting function used to generate the bottom ('Desirability') row of the chart, which depicts whether a variable correlates positively or negatively with RT-LAMP reaction time. FIG. 15A presents results for RT-LAMP amplification with Cas14a DETECTR in single reaction volume (one-pot). FIG. 15B shows a DETECTR assay screening buffers and alternative polymerases that enable RT-LAMP to work at lower temperatures (normally at 65C). FIG. 16 shows the layout of the LAMP plate used in an RT-LAMP assay screening buffers and alternative polymerases that enable RT-LAMP to work at lower temperatures (normally at 65C). FIGs. 17A-17B show assays evaluating the performance of Cas variants on a putative target site in SARS-CoV-2. FIG. 18 shows a strategy to simply DETECTR workflow for CRISPR diagnostics into a one-pot assay. FIGs. 19A-19B present results for HotPot involving LAMP amplification with Cas14a DETECTR in single reaction volume (one-pot). FIG. 20 presents results for identifying buffers that are compatible with Cas14a and low temperature RT-LAMP (LowLAMP). FIG. 21 presents results involving the impact of individual components on the performance of Cas14 at low temperature RT-LAMP conditions. FIG. 22 presents results for LAMP amplification with Cas 14a DETECTR in single reaction volume (one-pot). FIGs. 23A-23B presents results for one-pot Cas14 with LowLAMP at 50°C. FIG. 24 presents results for one-pot Cas14 with Bsm DNA polymerase at 55°C. FIG. 25 presents results for a limit of detection study involving one-pot DETECTR (HotPot). FIG. 26 presents results for a limit of detection study involving one-pot DETECTR (HotPot), where two different DNA polymerases at 55C were tested. FIG. 27 shows the results of using thermostable tracrRNA in the HotPot assays. FIG. 28 shows the results of using sgRNA in the HotPot assays. FIG. 29 presents results for a study involving replacing Bst polymerase in the NEAR assay, showing enablement for SARS-CoV-2 detection at lower temperatures. FIG. 30 presents results for NEAR assay amplification functions in Cas14a optimal buffers. FIG. 31 presents results for Cas14a functions in a range of KOAc salt concentrations. FIG. 32 presents results for a study involving increasing concentrations of KOAc to improve NEAR performance in Cas14a optimal buffers. FIG. 33 presents results for a study involving increasing concentrations of KOAc to improve NEAR performance in Cas14a optimal buffers. FIG. 34A - 34B present sequences and results for performance of Cas 14a.1 crRNAs on SARS-CoV-2 E-gene amplicon, respectively. FIG. 35 presents results for the evaluation of the performance of Klenow(exo-) NEAR assay in IB13 buffer at decreasing salt concentrations. FIG. 36 presents an overview of rolling circle amplification (sRCA) FIG. 37 presents results from screening dumbbell DNA templates for sRCA. FIG. 38 presents results from a study involving the ability of Cas14a to detect product of RCA reaction across increasing temperatures. FIG. 39 presents results from a study involving the effects of trigger oligos. FIG. 40 presents results from a study involving a titration of trigger oligos for Cas14 one-pot sRCA. FIG. 41 presents results from evaluating of a Cas12 variant (SEQ ID NO: 28) in one-pot sRCA. FIG. 42 presents an overview of RCA positive feedback for Cas13. FIG. 43 presents results from evaluating Cas13-compatible DNA templates for RCA. FIG. 44 presents results from a study evaluating whether a Cas13-compatible DNA template is functional in RCA. FIG. 45 presents results from a study involving Cas13 functionality in a one-pot sRCA reaction across increasing temperatures. FIG. 46 presents an overview of CasPin. FIG. 47 presents potential hairpin structures for CasPin. FIG. 48 presents results for an initial design using two hairpins. FIG. 49 presents a schematic of combined gRNA and reporter immobilization on the left and results for immobilization of DETECTR components using NHS-Amine chemistries on the right. FIG. 50 presents results from optimizing the conjugation buffer to reduce non-specific binding. FIG. 51 presents results from a study involving immobilizing different combinations of reporter + guide + a Cas12 variant (SEQ ID NO: 28). FIG. 52 presents results from a study optimizing gRNA and target concentrations to improve signal-to-noise ratio for immobilized DETECTR. FIGs. 53A-53B present modifications and results from evaluating various amino modifications for DETECTR immobilization, respectively. FIG. 54 presents results for the FASTR assay, involving detection of SARS-CoV-2 with rapid thermocycling + CRISPR Dx. FIG. 55 presents results from a study to determine top performing polymerases and buffers for the FASTR assay. FIG. 56 presents results for single copy detection of SARS-CoV-2 with FASTR. FIG. 57 presents results for variations on rapid cycling times for denaturation and annealing / extension in FASTR. FIG. 58 presents results for minimizing RT time for FASTR. FIG. 59 presents results for higher pH buffers that improve FASTR performance. FIG. 60 presents results for FASTR compatibility with crude lysis buffers. FIG. 61 presents results for non-optimized multiplexing of FASTR. FIG. 62 presents results for multiplex FASTR. FIG. 63 presents results for the limit of detection of multiplex FASTR. FIG. 64 presents key primers and gRNAs. FIG. 65 presents results depicting the enhancement of the signal generated in a Hotpot reaction with the addition of Thermostable Inorganic Pyrophosphatase (TIPP) in comparison to control conditions lacking TIPP, target RNA or both. FIG. 66A and FIG. 66B present results from DETECTR lateral flow Hotpot reaction assay strips, depicting the enhancement of the signal generated in a Hotpot reaction with the addition of Thermostable Inorganic Pyrophosphatase (TIPP) in comparison to control conditions lacking TIPP, target RNA or both. FIG. 67 shows an exemplary workflow for DETECTR-based HotPot reactions. FIG. 68 shows fluorescence results of HotPot reactions with reporters immobilized on glass beads. FIG. 69 shows lateral flow strip results using samples from the same experiments conducted to yield results illustrated in FIG. 68. FIG. 70 shows fluorescence results of HotPot reactions with reporters immobilized on magnetic beads. FIG. 71 shows fluorescence results of DETECTR-based HotPot assays for a variety of respiratory disease nucleic acid sequence targets. FIGS. 72A-72C shows results of limit of detection experiments for initial HotPot assay testing. FIG. 73 shows the fluorescence detected from HotPot assays in the presence of various additives. FIGS. 74A-74B shows the influence of select additives that increase the speed and / or the signal strength of some HotPot assays. FIG. 75 shows HotPot results from experiments conducted with various amounts of a few additives. FIG. 76 shows HotPot results from experiments conducted with various amounts of a few additives with BSM DNA Polymerases. FIGS. 77A-77B show lateral flow assay results of DETECTR-based OnePot and HotPot assays conducted with hydrogels comprising immobilized reporters. FIG. 78 shows an exemplary hydrogel comprising immobilized reporters copolymerized therein. FIGS. 79A and 79B show exemplary multiplexing strategies for hydrogel immobilized DETECTR systems. FIG. 80 shows a schematic of the NEAR reaction. A forward and reverse primer consisting of a nicking enzyme stabilization site and recognition region able amplify a target region of interest into a single-stranded DNA molecule. A guide RNA will bind a region complementary to the amplified ssDNA, allowing further detection by a DETECTR system. FIGS. 81A-81B show an exemplary NEAR-DETECTR reaction. FIG. 81A show a forward and reverse primer flanking the target region. In addition, a panel of 19 guide RNAs are shown in comparison to the amplicon. FIG. 81B shows the detection of the amplicon using NEAR-DETECTR using the above mentioned guide RNAs. FIGS. 82A-82B shows an example NEAR reaction with an exemplary guide RNA showing detection of the E-gene of SARS-CoV2. FIG. 82A shows an example NEAR reaction with forward and reverse NEAR primers with a guide RNA. FIG. 82B shows the NEAR-DETECTR reaction following amplification, showing the resulting signal of 20,000 copies or 0 copies of the amplicon in solution. FIG. 83 shows the resulting signal of a NEAR-DETECTR reaction in which the pre-amplification time is varied prior to DETECTR. FIG. 84 shows the comparison of the NEAR-DETECTR reaction using orthogonal Cas systems (Cas12 Variant (SEQ ID NO: 28), Cas13 Variant (SEQ ID NO: 154), and Cas14 Variant (SEQ ID NO. 63)). The experiments were performed in the presence or absence of the target NEAR amplicon, showing different cleavage preferences for the reporter molecule. FIG. 85 shows the optimization of the NEAR reaction using different magnesium (Mg2+) concentrations. This shows the time to result (in minutes) comparing different buffer compositions and different concentrations of added magnesium. The top panel shows the following conditions using Bst2.0, and the bottom panel shows the following experimental conditions using Bst.3.0. These results informed the following experimental conditions for the NEAR reaction: Bst2.0, 12 mM Mg2+, at 60C, resulting in a less than 5 minute amplification time and approximately 20,000 copies. FIGS. 86A-86B shows the experimental design of the primer and guide RNA design and the results of these designs. In FIG. 86A, eight primer pairs (R1763 F(1-8) / R(1-8)) and the guide RNA R1763. In FIG. 86B, the aforementioned primer pairs and guides R1763 and R1765 are used in a NEAR-DETECTR reaction to determine the efficacy of the reaction using different primer pairs. The raw fluorescence (AU) of the NEAR-DETECTR reaction is reported. FIGS. 87A-87B describes the optimization of the hinge stabilization region in the NEAR primers to determine if the nicking enzyme activity can be modulated. In FIG. 87A, the hinge stabilization loop region is modified to alter the melting temperature of the stem loop. In FIG. 87B, the modified stem loops as shown in FIG. 87A were used for detection of the SARS-CoV-2 E-gene using different inputs of amplicons resulting from a NEAR amplification (1,000, 500, and 200 input amplicon copies). FIG. 88 shows a comparison of a reverse transcription-NEAR-DETECTR (RT-NECTR) reaction using different reverse transcriptases. Wartmstart RTx (NEB), Bst 3.0, and Omniscript RT (Qiagen) were used on different amounts of input RNA in order to compare the limit of detection (LOD) of these reaction conditions. FIG. 89 shows the experimental results of an RT-NECTR reaction using differing concentrations of NEAR primers. FIG. 90 shows the LOD of the E-gene of SARS-COV-2 using RT-NECTR using different amplification times and input copies for the RT-NECTR reaction. FIG. 91 shows detection of the SARS-COV-2 E gene using different Cas systems (Cas12 Variant (SEQ ID NO: 28), Cas13 Variant (SEQ ID NO: 154), and Cas14 Variant (SEQ ID NO. 63)) and a panel of different guides. DETAILED DESCRIPTION

[0027] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to encompass "and / or" unless otherwise stated.

[0028] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "at least," "greater than" or "greater than or equal to" applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0029] Whenever the term "no more than," "less than," "less than or equal to," or "at most" precedes the first numerical value in a series of two or more numerical values, the term "no more than," "less than," "less than or equal to," or "at most" applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0030] Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0031] The present disclosure provides various compositions, methods, and devices of use thereof for assaying and detecting a nucleic acid. In many cases, nucleic acid detection comprises multiple steps (e.g., multiple reactions). For example, nucleic acid detection often comprises amplification of a target nucleic acid followed by detection. Augmenting the challenge of nucleic acid detection, multiple steps often require different conditions, which can necessitate buffer exchanges or sample transfers that increase the potential for contamination and sample loss. Furthermore, many single-buffer systems only support a single type of reaction, and drastically retard the rates of other reactions performed within them.

[0032] To address these challenges, aspects of the present disclosure provide buffers, systems, and compositions that can support multiple nucleic acid detection steps (e.g., multiple types of reactions) with optimal or close to optimal rates. Such buffers, systems, and compositions can enable nucleic acid detection without buffer exchanges or sample transfers, thereby diminishing total assay times, decreasing sample loss and contamination, and minimizing user input.

[0033] For example, particular aspects of the present disclosure provide containers comprising all of the reactants necessary for detection of a target nucleic acid from a sample. In such cases, no further user steps may be required once the sample is inserted into the container (e.g., through a pierceable film, membrane, or septum) and the container is placed within an instrument (e.g., a fluorimeter comprising a sample heater). In some aspects, a plurality of containers may be provided in a kit or on a single support (e.g., each container is a well on a multi-well plate). Two containers among the plurality of containers may comprise reagents targeting different nucleic acid sequences for detection to enable multiplexing.

[0034] 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 detector nucleic acid). In general, the term "reporter" as used in this context refers to a reagent comprising a polynucleotide, wherein cleavage of the polynucleotide results in a change in a signal. For example, the reporter may comprise a fluorescent label joined to a quencher by a short polynucleotide sequence. Little to no fluorescence is detectable from the fluorescent label when joined to the quencher. However, upon cleavage of the polynucleotide, the fluorescent label is separated from the quencher, resulting in a significant and detectable increase in fluorescent signal upon excitation of the label. Alternative labels and arrangements for producing a change in signal upon cleavage of the polynucleotide portion of the reporter are possible, and illustrative examples are described herein. The polynucleotide of the reporter can comprise DNA, RNA, modified nucleotides, or a combination of two or more of these. 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.

[0035] 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 a CRISPR-based programmable nuclease.

[0036] Various methods, reagents, compositions, systems, and devices disclosed herein use a programmable nuclease complexed with guide nucleic acid sequence to detect the presence or absence of, and / or quantify the amount of, a target nucleic acid sequence. Binding of a guide nucleic acid with a target nucleic acid may activate a programmable nuclease to cleave single stranded nucleotides in a sequence non-specific manner, hereinafter referred to as "transcollateral" cleavage or "trans cleavage". Some assays of the present disclosure detect single-stranded, non-target nucleic acid cleavage to determine the presence and / or quantity of a target nucleic acid. 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. Herein, detection of reporter cleavage (directly or indirectly) to determine the presence of a target nucleic acid sequence may be referred to as 'DETECTR'. In some embodiments, described herein is a method of assaying for a target nucleic acid in a sample comprising contacting the target nucleic acid with a programmable nuclease, a non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, and a reporter, and assaying for a change in a signal, wherein the change in the signal is produced by or indicative of cleavage of the reporter.

[0037] Assays disclosed herein may comprise amplification (e.g., loop-mediated amplification (LAMP) or recombinase polymerase amplification (RPA), rolling circle amplification (RCA), nicking enzyme amplification reaction (NEAR), etc.) of a target nucleic acid sequence (e.g., a viral nucleic acid extracted from a patient). A target nucleic acid may also be reverse transcribed. An assay targeting an RNA sequence may utilize reverse transcription (RT) and amplification to generate amplicons for a programmable nuclease-based detection (e.g., DETECTR) reaction. A target nucleic acid may be amplified by thermal amplification (e.g., PCR) or isothermal amplification (e.g., LAMP, RCA, NEAR). An assay may utilize an amplification reaction and a programmable nuclease-based detection (e.g., DETECTR) reaction.

[0038] Among the various aspects of the present disclosure are compositions and methods for rapid and accurate detection of a target nucleic acid. In many cases, target nucleic acids are present in low copy numbers or as small proportions of the total nucleic acid content from a sample. This is a particular challenge in pathogen (e.g., viral) diagnostics, as nucleic acid biomarkers are often present in copy numbers below 1000 in samples derived from patients. Owing to this challenge, nucleic acid detection typically requires multi-step and multi-reaction assays. However, the assays themselves often have strict and non-overlapping requirements for the physical and chemical conditions in which they can be performed. For example, conditions that support rapid amplification reactions are often unsuitable for programmable nuclease-mediated reactions (e.g., CRISPR-Cas enzyme-mediated reactions), such as DETECTR reactions. Thus, nucleic acid detection methods frequently require sample transfers and buffer exchanges, resulting in long assay times leading to low yields.

[0039] In various aspects, the present disclosure provides a range of compositions that facilitate rapid, single buffer nucleic acid detection assays. Among such compositions, the present disclosure provides buffers capable of supporting rapid amplification and programmable nuclease (e.g., CRISPR-Cas) enzyme mediated reactions (e.g., DETECTR reactions). In such cases, the buffer may not only enable fast reaction rates, but also high reaction yields. Furthermore, multi-reaction buffer compatibility can drastically diminish an assay's required user input. Further aspects of the disclosure build on these favorable characteristics, and provide methods for performing single buffer nucleic acid detection assays, as well as kits for performing such assays.Assaying Methods

[0040] In some embodiments, a programmable nuclease can be used for detection of a target nucleic acid in a sample from a subject. The programmable nuclease may be provided in a buffer which enables fast (e.g., within 25%, 50%, or 75% of the fastest reported reaction rate for the programmable nuclease at a particular temperature) kinetics for the programmable nuclease and for reagents for other reactions (e.g., amplification, viral or cellular lysis, nucleic acid digestion, etc.). This can be particularly advantageous for low stability targets, such as RNA, which can be prone to rapid degradation, precipitation, denaturation, or side reactions. Whereas detecting a nucleic acid from a low titer sample (e.g., a sample comprising 1000, 500, 100, 50, 25, or 10 target nucleic acid molecules) may be unfeasible if a sample needs to be transferred between multiple reaction volumes, a single-buffer reaction system can enable detection of sparse nucleic acid targets by minimizing sample loss between steps.

[0041] The target nucleic acid may comprise a sequence associated with a pathogen (e.g., a virus), a human gene of interest (e.g., an oncogene such as BRCA1), a nucleic acid sequence for uniquely identifying an individual, or a fungal or bacterial nucleic acid sequence (e.g., for assessing skin health or a gut microbiome). For example, a programmable nuclease can be complexed with a guide nucleic acid that hybridizes to a target sequence of a target nucleic acid from coronavirus. The complex can be contacted to a sample from a subject. The target nucleic acid may or may not be present in the sample. If present, the target nucleic acid in the sample can optionally be reverse transcribed (RT). The target nucleic acid can be amplified by thermal amplification (e.g., PCR, FASTR) or isothermal amplification (e.g., LAMP, RPA, RT-RPA, or RT-LAMP). In some embodiments, reverse transcription and isothermal amplification may be performed simultaneously.

[0042] Upon activation, the programmable nuclease can cleave a reporter, which may comprise a detectable label attached to a polynucleotide (e.g., polydeoxyribonucleotide or polyribonucleotide). In some embodiments of the assay, upon cleavage of the polynucleotide, the detectable label emits a detectable signal, which is then detected and quantified (e.g., the detectable label may be a fluorophore and the detectable signal may be fluorescence). Upon detection of the detectable signal, it can be determined that the sample from the subject contained a target nucleic acid. In some embodiments, a programmable nuclease-based detection assay may detect multiple target nucleic acids or amplicons. For example, a programmable nuclease-based detection assay may target multiple non-overlapping or partially overlapping portions of a sequence of interest, while an amplification reaction may tile primers over a region of a target nucleic acid. Additionally, a programmable nuclease-based detection assay and / or amplification reaction may target multiple distinct sequences, such as sequences from separate portions of a genome or from separate genomes.

[0043] The compositions and methods of use thereof disclosed herein include using a programmable nuclease such as a Cas12 protein, a CasΦ protein, Cas14 protein, or a Cas13 protein to assay for, detect, and / or quantify a target nucleic acid. In some embodiments, a Cas12 protein, Cas13 protein, Cas14 protein, or a CasΦ protein is used for detection of a target nucleic acid in a sample from a subject. For use in an assay with a Cas12 protein, CasΦ protein, Cas14 protein, or a Cas13 protein, a target nucleic acid in a sample can be reverse transcribed and amplified by thermal (e.g., PCR, FASTR) or isothermal amplification (e.g., LAMP, RCA, NEAR). For use in an assay with a Cas13 protein, the amplified target nucleic acids can be transcribed back into RNA. If the subject is infected with coronavirus, the guide nucleic acid hybridizes to the target nucleic acid or amplicon thereof leading to activation of the Cas12 protein, Cas14 protein, CasΦ protein, or Cas13 protein. Upon activation, the Cas12 protein, the CasΦ protein, the Cas14 protein, or the Cas13 protein can cleave a reporter, wherein the reporter comprises a detectable label attached to the nucleic acid for cleavage by a Cas12 protein, the Cas14 protein a Cas13 protein, or a CasΦ protein. In some embodiments of the assay, upon cleavage of the reporter, the detectable label emits a detectable signal, which can then be captured and quantified (e.g., the detectable label may be a fluorophore and the detectable signal may be fluorescence). Upon detection of a detectable label, it can be determined that the sample comprised the target nucleic acid or target nucleic acids.

[0044] In some embodiments, a programmable nuclease having at least 60% sequence identity to SEQ ID NO: 18-170, 221-268, or 397-423 can be used for detection of a target nucleic acid (e.g., from a coronavirus such as SARS-CoV-2) in a sample from a subject. For example, a programmable nuclease having at least 60% sequence identity to SEQ ID NO: 18 can be complexed with a guide nucleic acid that hybridizes to a target sequence of a target nucleic acid from coronavirus. For example, a programmable nuclease having at least 60% sequence identity to any one of SEQ ID NOs: 397 , 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, or 423 can be complexed with a guide nucleic acid that hybridizes to a target sequence of a target nucleic acid from coronavirus. For example, a programmable nuclease having at least 60% sequence identity to SEQ ID NO: 406 can be complexed with a guide nucleic acid that hybridizes to a target sequence of a target nucleic acid from coronavirus.

[0045] Also described herein are methods, reagents, and devices for detecting the presence of a target nucleic acid in a sample. The methods, reagents, and devices for detecting the presence of a target nucleic acid in a sample can be used in rapid lab tests for detection of a target nucleic acid of interest (e.g., target nucleic acids from a target population). In particular, provided herein are methods, reagents, and devices wherein the rapid lab tests can be performed in a single system. The target nucleic acid may be a portion of a nucleic acid from a virus (e.g., coronavirus) or other agents responsible for a disease in the sample.

[0046] In some embodiments, programmable nucleases disclosed herein are activated by RNA or DNA to initiate trans cleavage activity of a reporter. A programmable nuclease as disclosed herein, in some cases, binds to a target RNA to initiate trans cleavage of a reporter, and this programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, a programmable nuclease as disclosed herein binds to a target DNA to initiate trans cleavage of a reporter, and this programmable nuclease can be referred to as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein is capable of being activated by a target RNA or a target DNA. For example, a Cas13 protein, such as a Cas13a, disclosed herein is activated by a target RNA nucleic acid or a target DNA nucleic acid to transcollaterally cleave an RNA reporter. In some embodiments, the Cas13 binds to a target ssDNA which initiates trans cleavage of an RNA reporter.

[0047] The detection of the target nucleic acid in the sample may indicate the presence of the disease in the sample and may 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. The detection of the target nucleic acid in the sample may indicate the presence of a disease mutation, such as a single nucleotide polymorphism (SNP) that provide antibiotic resistance to a disease-causing bacteria. The detection of the target nucleic acid is facilitated by a programmable nuclease. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity, which can also be referred to as "collateral" or "transcollateral" cleavage.

[0048] Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of reporters with a detection moiety. Once the reporter is cleaved by the activated programmable nuclease, the detection moiety is released from the reporter and generates a detectable signal. Often the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid. Sometimes the detection moiety binds to a capture molecule on the support medium to be immobilized. The detectable signal can be visualized on the support medium to assess the presence or level of the target nucleic acid associated with an ailment, such as a disease. 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. These assays, which leverage the transcollateral cleavage properties of programmable nucleases (e.g., CRISPR-Cas enzymes) are often referred to herein as DNA endonuclease targeted CRISPR trans reporter (DETECTR) reactions. A programmable nuclease-based detection (e.g., DETECTR) reaction can be performed in a fluidic device.

[0049] In some embodiments, the present disclosure provides for Cas12 detection of a target nucleic acid from a coronavirus. In this case, nucleic acids (e.g., RNA) from a sample may be optionally reverse transcribed and / or amplified into DNA. Any Cas12 protein disclosed herein may be complexed with a guide nucleic acid designed to hybridize to a nucleic acid sequence of the (optionally reverse transcribed and / or amplified) DNA. DETECTR reactions can then be carried out. In the presence of reverse transcribed and amplified DNA indicative of coronavirus, Cas12 is activated to transcollaterally cleave a reporter, emitting a detectable signal (e.g., fluorescence). In some embodiments, the present disclosure provides for Cas13 detection of a target nucleic acid from a coronavirus. In this case, RNA in a sample may be either directly detected by complexing a Cas13 enzyme with a guide nucleic acid designed to hybridize to a target RNA sequence from a coronavirus or, RNA may be reverse transcribed, amplified, and in vitro transcribed prior to contacting it with a Cas13 enzyme complexed with a guide nucleic acid designed to hybridize this amplified target RNA sequence from a coronavirus. In the presence of the RNA (unamplified or amplified), Cas13 may be activated to transcollaterally cleave a reporter, thereby emitting a detectable signal (e.g., fluorescence). In some embodiments, the present disclosure provides for Cas14 detection of a target nucleic acid from a coronavirus. In this case, nucleic acids (e.g., RNA) from a sample may be optionally reverse transcribed and / or amplified into DNA. Any Cas14 protein disclosed herein may be complexed with a guide nucleic acid designed to hybridize to a nucleic acid sequence of the (optionally reverse transcribed and / or amplified) DNA. DETECTR reactions can then be carried out. In the presence of reverse transcribed and amplified DNA indicative of coronavirus, Cas14 is activated to transcollaterally cleave a reporter, emitting a detectable signal (e.g., fluorescence). In some embodiments, the present disclosure provides for CasPhi detection of a target nucleic acid from a coronavirus. In this case, nucleic acids (e.g., RNA) from a sample may be optionally reverse transcribed and / or amplified into DNA. Any CasPhi protein disclosed herein may be complexed with a guide nucleic acid designed to hybridize to a nucleic acid sequence of the (optionally reverse transcribed and / or amplified) DNA. DETECTR reactions can then be carried out. In the presence of reverse transcribed and amplified DNA indicative of coronavirus, CasPhi is activated to transcollaterally cleave a reporter, emitting a detectable signal (e.g., fluorescence).

[0050] Also described herein is a kit for detecting a target nucleic acid (e.g., from a coronavirus such as SARS-CoV-2). The kit may comprise a support medium; a guide nucleic acid sequences targeted to a target nucleic acid sequence; a programmable nuclease capable of being activated when complexed with a guide nucleic acid and a target nucleic acid; and a single-stranded reporter comprising a detection moiety, wherein the reporter is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal. In some cases, the target nucleic acid for detecting a coronavirus may comprise a sequence with at least 60% sequence similarity to that of SEQ ID NOs: 179-184. In some cases, the guide nucleic acid for detection of a coronavirus may comprise a sequence with at least 60% sequence similarity to the sequences in SEQ ID NOs: 318-327.

[0051] A biological sample from an individual or an environmental sample can be tested for the presence of a particular nucleic acid sequence (e.g., whether a human sample comprises coronavirus). The detection of the target nucleic acid detected can also indicate that one or more of the target populations is wild-type or comprises a mutation, such as a mutation that confers resistance to treatment, such as antibiotic treatment. A sample from an individual or from an environment is applied to the reagents described herein. If the target nucleic acid is present in the sample, the target nucleic acid binds to the guide nucleic acid to activate the programmable nuclease. The activated programmable nuclease cleaves the reporter and generates a detectable signal that can be visualized, for example on a support medium. If the target nucleic acid is absent in the sample or below the threshold of detection, the guide nucleic acid remains unbound, the programmable nuclease remains inactivated, and the reporter remains uncleaved. Such methods, reagents, and devices described herein may allow for detection of target nucleic acid, and in turn the disease associated with the target nucleic acids (e.g., coronavirus such as SARS-CoV-2), in remote regions or low resource settings without specialized equipment. Also, such methods, reagents, and devices described herein may allow for detection of target nucleic acid, and in turn the disease associated with the target nucleic acids, in healthcare clinics or doctor offices without specialized equipment. In some cases, this provides a point of care testing for users to quickly and easily test for a disease or infection with high sensitivity at home or in an office of a healthcare provider. Assays that deliver results in under an hour, for example, in 15 to 60 minutes, are particularly desirable for at home testing for many reasons. For example, antivirals can be most effective when administered within the first 48 hours after disease exposure. Thus, the methods disclosed herein, which are capable of delivering results in under an hour, may allow for the delivery of anti-viral therapy during the first 48 hours after infection. Additionally, the systems and assays provided herein, which are capable of delivering quick diagnoses and results, can help keep or send a patient at home, improve comprehensive disease surveillance, and prevent the spread of an infection. In other cases, this provides a test, which can be used in a lab to detect one or more nucleic acid populations or varieties of interest in a sample from a subject. In particular, provided herein are methods, reagents, and devices, wherein the high sensitivity lab tests can be performed in a single assay. In some cases, this may be valuable in detecting diseases in a developing country and as a global healthcare tool to detect the spread of a disease or efficacy of a treatment or provide early detection of a disease.

[0052] Some methods as described herein use an editing technique, such as a technique using an editing enzyme or a programmable nuclease and guide nucleic acid, to detect a target nucleic acid. An editing enzyme or a programmable nuclease in the editing technique can be activated by one or more target nucleic acids, after which the activated editing enzyme or activated programmable nuclease can cleave nearby single-stranded nucleic acids, such reporters with a detection moiety. A target nucleic acid can be amplified by isothermal amplification or thermocycling amplification and then an editing technique can be used to detect the marker. In some instances, the editing technique can comprise an editing enzyme or programmable nuclease that, when activated, cleaves nearby RNA or DNA as the readout of the detection. The methods as described herein in some instances comprise obtaining a cell-free DNA sample, amplifying DNA from the sample, using an editing technique to cleave reporters, and reading the output of the editing technique. In other instances, the method comprises obtaining a fluid sample from a patient, and without amplifying a nucleic acid of the fluid sample, using an editing technique to cleave reporters, and detecting the nucleic acid. The method can also comprise using single-stranded detector DNA, cleaving the single-stranded detector DNA using an activated editing enzyme, wherein the editing enzyme cleaves at least 50% of a population of single-stranded detector DNA as measured by a change in color. A number of samples, guide nucleic acids, programmable nucleases or editing enzymes, support mediums, target nucleic acids, single-stranded reporters, and reagents are consistent with the devices, systems, fluidic devices, kits, and methods disclosed herein.

[0053] Also disclosed herein are reporters and methods detecting a target nucleic using the reporters. Often, the reporter is a protein-nucleic acid. For example, a method of assaying for a target nucleic acid in a sample comprises contacting the sample to a plurality of complexes comprising a guide nucleic acid, each guide nucleic acid sequence comprising a segment that is reverse complementary to a segment of a target nucleic acid sequence within a target nucleic acid population and programmable nucleases that exhibits sequence independent cleavage upon forming complexes 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 protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of one or more of the target nucleic acid populations in the sample and wherein absence of the signal indicates an absence of the target nucleic acid population in the sample. Often, the protein-nucleic acid is an enzyme-nucleic acid or an enzyme substrate-nucleic acid. The nucleic acid can be DNA, RNA, or a DNA / RNA hybrid. The methods described herein use a programmable nuclease, such as the CRISPR / Cas system, to detect a target nucleic acid (e.g. from a coronavirus such as SARS-CoV-2). A method of assaying for a target nucleic acid (e.g. from a coronavirus such as SARS-CoV-2) in a sample, for example, comprises: a) contacting the sample to a plurality of complexes comprising a guide nucleic acid, each guide nucleic acid sequence comprising a segment that is reverse complementary to a segment of a nucleic acid target sequence within a target nucleic acid population, and programmable nucleases that exhibits sequence independent cleavage upon forming complexes comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; b) contacting the complexes to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, wherein the signal indicates a presence of one or more of the target nucleic acid populations in the sample and wherein absence of the signal indicates an absence of the target nucleic acid population in the sample. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.

[0054] Cleavage of the protein-nucleic acid produces a signal. For example, cleavage of the protein-nucleic acid produces a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. Various devices can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample.

[0055] The present disclosure provides buffers capable of supporting rapid amplification and programmable nuclease-based reactions (e.g., DETECTR).

[0056] In some assays, such a buffer comprising amplification and programmable nuclease-based detection reagents is contacted to a sample, thereby enabling simultaneous amplification and programmable nuclease-based detection reactions. For example, a buccal swab sample may be added to a buffer comprising amplification and programmable nuclease-based detection reagents targeting a particular oral bacterium gene, thereby enabling an assay for that particular oral bacterium. In some assays, a target nucleic acid is first contacted with amplification reagents, and later (e.g., after the amplification reaction has come to completion) contacted with reagents for a programmable nuclease-based detection reaction. In some assays, a target nucleic acid is first contacted with amplification reagents, and later sequentially contacted with different sets of programmable nuclease-based detection reagents targeting different nucleic acid sequences, wherein programmable nuclease-based detection reagent additions that lead to an increase in signal (e.g., fluorescence signal) may indicate the presence of a particular nucleic acid sequence.Sample

[0057] A number of samples are consistent with the methods, reagents, and devices disclosed herein.

[0058] These samples can comprise a target nucleic acid for detection of an ailment, such as a disease, pathogen, or virus, such as influenza. Generally, a sample from an individual or an animal or an environmental sample can be obtained to test for presence of a disease, or any mutation of interest. A biological sample from the individual may be blood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastric secretions, nasal secretions, sputum, pharyngeal exudates, urethral or vaginal secretions, an exudate, an effusion, or tissue. A tissue sample may be dissociated or liquified prior to application to the detection system of the present disclosure. A sample from an environment may 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 or be applied neat to the detection system. Sometimes, the sample is contained in no more 20 µL. The sample, in some cases, is contained in no more than 1, 5, 10, 15, 20, 25, 30, 35 40, 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. Sometimes, the sample is contained in more than 500 µL. The sample may be contained within a solid, membranous, or mesh material, such as a swab (e.g., a buccal or nasal swab).

[0059] In some instances, the sample is taken from single-cell eukaryotic organisms; a plant or a plant cell; an algal cell; a fungal cell; 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 comprises 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 comprises nucleic acids expressed from a cell.

[0060] The sample used for disease testing may comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein. A portion of a nucleic acid can be from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA. A portion of 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 portion of 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 sequence can be reverse complementary to a guide nucleic acid. Each target sequence of the multiple target sequences can be reverse complementary to a distinct guide nucleic acid.

[0061] 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 nanoliters (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, 35 40, 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.

[0062] 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.

[0063] 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.

[0064] 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); 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 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, 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.

[0065] 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 contribute 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.

[0066] 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, 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.

[0067] The method can comprise generating one or more droplets, aliquots, or subsamples from the sample. The one or more droplets, aliquots, or subsamples can correspond to a volumetric portion of the sample. The sample can be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more droplets, aliquots, or subsamples. In some embodiments, the sample is not divided into subsamples. As used herein, a droplet can refer to a volumetric portion of the sample, a partitioned sub-sample of the sample, and / or an aliquot of the sample. In some cases, the detection chamber is configured to receive and contact the plurality of droplets with at least one programmable nuclease disposed on a surface of said detection chamber. The at least one programmable nuclease can comprise a guide nucleic acid complexed with a programmable nuclease. In some cases, the programmable nuclease 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 complexes comprising different guide RNAs.

[0068] The method can comprise amplifying one or more targets within each droplet, aliquot, or subsample. Amplification of the one or more targets within each droplet can be performed in parallel and / or simultaneously for each droplet. Dividing the sample into a plurality of droplets can enhance a speed and / or an efficiency of the amplification process (e.g., a thermocycling process) since the droplets comprise a smaller volume of material than the bulk sample introduced. Amplifying the one or more targets within each individual droplet can also permit effective amplification of various target nucleic acids that cannot be amplified as efficiently in a bulk sample containing the various target nucleic acids if the bulk sample were to undergo a singular amplification process. In some embodiments, amplification is performed on the bulk sample without first dividing the sample into subsamples.The method can further comprise using a CRISPR-based or programmable nuclease-based detection module to detect one or more targets (e.g., target sequences or target nucleic acids) in the sample. In some cases, the sample can be divided into a plurality of droplets, aliquots, or subsamples to facilitate sample preparation.

[0069] Described herein are various methods of sample preparation and reagent storage. Any of the devices described herein may comprise one or more sample preparation reagents. Any of the devices 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, as claimed. 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 lyophilized 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.

[0070] 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, and / or base / acid lysis.

[0071] 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).

[0072] In some embodiments, lysis of a collected sample can be performed, as claimed. Lysis can be performed using a protease (e.g., a Proteinase K or PK enzyme). 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.

[0073] 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). In some cases, a heating element integrated into the 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 detection device.

[0074] In some cases, a target nucleic acid within the sample can undergo amplification before binding to a guide nucleic acid, for example a crRNA or sgRNA of a CRISPR enzyme, as claimed. 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), rolling circle amplification (RCA), nicking enzyme amplification reaction (NEAR), FASTR, and / or polymerase chain reaction (PCR). In some instances, digital droplet amplification can used. Such nucleic acid amplification of the sample can improve at least one of a sensitivity, specificity, or accuracy of the detection of the target nucleic acid. 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, 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.

[0075] In some embodiments, amplification can comprise thermocycling of the sample. Thermocycling can be carried out for one or more droplets of the sample in parallel and / or independently in separate locations. This can be accomplished by methods such as (1) by holding droplets stationary in locations where a heating element is in close proximity to the droplet on one of the droplet sides and a heat sink element is in close proximity to the other side of the droplet, or (2) flowing the droplet through zones in a fluid channel where heat flows across it from a heating source to a heat sink. In some cases, one or more resistive heating elements can be used to perform thermocycling. In some cases, the thermocyling of the sample may comprise one or more reactions at different temperatures. In some cases, the reactions can include one or more reactions selected from an annealing reaction, a denaturation reaction, an extension reaction, reverse transcription reaction, and a detection reaction. In some cases, the thermocycling of the sample may comprise amplification of one or more nucleic acids in the sample.

[0076] In some cases, the annealing temperature of the reaction is performed at a temperature around 45°C to 75°C. In some embodiments, the annealing temperature may be at a temperature of about 45 °C, about 47 °C, about 48 °C, about 49 °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, or about 76 °C.

[0077] In some cases, the denaturation temperature of the reaction is performed at a temperature around 90°C to about 110°C. In some embodiments, the denaturation temperature may be at a temperature of about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C, about 100°C, about 101°C, about 102°C, about 103°C, about 104°C, about 105°C, about 106°C, about 107°C, about 108°C, about 109°C, or about 110°C.

[0078] In some cases, the extension temperature of the reaction is performed at a temperature from around 55°C to about 85°C. In some embodiments, the extension temperature may be at a temperature of about 55°C, about 57°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 68°C, about 70°C, about 71°C, about 72°C, about 73°C, about 75°C, about 76°C, about 78°C, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, or about 85°C.

[0079] In some cases, the reverse transcription step of the reaction is performed at a temperature of around 45°C to about 75°C. In some embodiments, the reverse transcription may be at a temperature of about 45°C, about 47°C, about 48°C, about 49°C, about 50°C, about 52°C, about 54°C, about 55°C, about 57°C, about 59°C, about 60°C, about 61°C, about 63°C, about 65°C, about 66°C, about 68°C, about 70°C, about 72°C, about 73°C, or about 75°C.

[0080] In some cases, the detection step of the reaction is performed at a temperature of about 30°C to about 50°C. In some embodiments, the detection step may be at a temperature of about 30°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, or about 50°C.

[0081] Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45°C. The nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. The nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some cases, the nucleic acid amplification reaction is performed at a temperature of from 20°C to 45°C, from 25°C to 40°C, from 30°C to 40°C, or from 35°C to 40°C. In some cases, the nucleic acid amplification reaction is performed at a temperature of from 45°C to 65°C, from 50°C to 65°C, from 55°C to 65°C, or from 60°C to 65°C. 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.

[0082] Additionally, target nucleic acid can optionally be amplified before binding to the guide nucleic acid (e.g., crRNA or sgRNA) of the programmable nuclease (e.g., CRISPR enzyme). This amplification can be PCR amplification or isothermal amplification. This nucleic acid amplification of the sample can improve at least one of sensitivity, specificity, or accuracy of the detection the target nucleic acid. The reagents for nucleic acid amplification can comprise a recombinase, a 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, 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 reaction is performed at a temperature of around 20-45°C. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 45-65 °C. The nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. The nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.

[0083] Devices of the present disclosure can be configured to perform droplet digitization or droplet generation. Droplet digitization or generation can comprise splitting a volume of the sample into multiple droplets, aliquots, or subsamples. The sample can have a volume that ranges from about 10 microliters to about 500 microliters. The plurality of droplets, aliquots, or subsamples can have a volume that ranges from about 0.01 microliters to about 100 microliters. The plurality of droplets, aliquots, or subsamples can have a same or substantially similar volume. In some cases, the plurality of droplets, aliquots, or subsamples can have different volumes. In some cases, the droplets, aliquots, or subsamples can be generated using a physical filter or one or more movable mechanisms (e.g., valves, etc.). In some cases, each droplet of the sample can undergo one or more sample preparation steps (e.g., nucleic acid purification, lysis, heat inactivation, amplification, etc.) independently and / or in parallel while the droplets are physically constrained or thermally isolated between two movable mechanisms.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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).

[0088] 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.

[0089] 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.

[0090] 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 10 1< non-target nucleic acids, 10 2< non-target nucleic acids, 10 3< non-target nucleic acids, 10 4< non-target nucleic acids, f, 10 6< non-target nucleic acids, 10 7< non-target nucleic acids, 10 8< non-target nucleic acids, 10 9< non-target nucleic acids, or 10 10< non-target nucleic acids.

[0091] In some cases, the target nucleic acid is present at less than 1 µg / ml in a sample. In some cases, the target nucleic acid is present at less than 100 ng / ml in a sample. In some cases, the target nucleic acid is present at less than 10 ng / ml in a sample. In some cases, the target nucleic acid is present at less than 1 ng / ml in a sample. In some cases, the target nucleic acid is present at less than 100 pg / ml in a sample. In some cases, the target nucleic acid is present at less than 10 pg / ml in a sample. In some cases, the target nucleic acid is present at less than 1 pg / ml in a sample. In some cases, the target nucleic acid is present at less than 100 fg / ml in a sample. In some cases, the target nucleic acid is present at less than 10 fg / ml in a sample. In some cases, the target nucleic acid is present at less than 1 fg / ml in a sample.

[0092] 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 10 1< non-target nucleic acids, 10 2< non-target nucleic acids, 10 3< non-target nucleic acids, 10 4< non-target nucleic acids, 10 5< non-target nucleic acids, 10 6< non-target nucleic acids, 10 7< non-target nucleic acids, 10 8< non-target nucleic acids, 10 9< non-target nucleic acids, or 10 10< non-target nucleic acids. The target nucleic acid populations can be present at different concentrations or amounts in the sample.

[0093] The compositions and methods of the present disclosure are compatible with intact and damaged nucleic acids. The target nucleic acid may be fragmented prior to amplification and / or detection (e.g., by a DETECTR assay). The target nucleic acid may comprise ordered or random fragmentation. The target nucleic acid may also comprise damage, such as oxidation or cross-linking. The target nucleic acid may comprise chemical, light-induced, and / or enzymatic cleavage. Conversely, the target nucleic acid may be unfragmented. A target nucleic acid sequence may be embedded within a longer nucleic acid. For example, a target nucleic acid may be a gene within an intact chromosome. A target nucleic acid may comprise a chemical modification, such as acetylation or methylation. A target nucleic acid may comprise a defined secondary, tertiary, or quaternary structure. A target nucleic acid may be complexed with another species, such as a histone.

[0094] Any of the above disclosed samples are consistent with the systems, assays, and programmable nucleases disclosed herein and can be used as a companion diagnostic with any of the diseases disclosed herein (e.g., a coronavirus infection), or can be used in reagent kits, point-of-care diagnostics, or over-the-counter diagnostics.Reagents

[0095] A number of reagents are consistent with the methods, reagents, and devices disclosed herein. Many aspects of the present disclosure provide buffers, systems, and compositions that enable fast kinetics (e.g., within 25%, 50%, 75%, or 90% of the fastest reported rate for a reaction at a given temperature, or coming to completion within one hour) for multiple types of reactions or processes. Such buffers, systems, and compositions enable multiple reactions to be performed on a sample without intervening sample transfers, buffer exchanges, or reagent removal. In some cases, reagents (e.g., enzymes) for multiple reactions may be provided in a single solution. Such cases may require no more than sample addition to determine whether a nucleic acid (e.g., a particular nucleic acid sequence) is present in the sample.

[0096] These reagents are compatible with the samples, methods, and devices as described herein for detection of an ailment, such as a disease. The reagents described herein for detecting a disease, such as coronavirus, comprise multiple guide nucleic acids, each guide nucleic acid targeting a target nucleic acid segment indicative of the disease. Each guide nucleic acid binds to the target nucleic acid comprising a segment of a nucleic acid sequence (e.g., a nucleic acid from coronavirus) as described herein. Each guide nucleic acid can bind to the target nucleic acid comprising a portion of a nucleic acid (e.g., a target nucleic acid from coronavirus) as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), that can confer resistance to a treatment, such as antibiotic treatment. Each guide nucleic acid binds to the target nucleic acid comprising a portion of a nucleic acid. Each guide nucleic acid is complementary to a target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid may be a RNA, DNA, or synthetic nucleic acids.

[0097] Disclosed herein are methods of assaying for a plurality of target nucleic acids (e.g., a plurality of nucleic acids from coronavirus) as described herein. For example, a method of assaying for a plurality of target nucleic acids in a sample comprises contacting the sample to a complex comprising a plurality guide nucleic acid sequences, each guide nucleic acid sequence comprising a segment that is reverse complementary to a segment of the target nucleic acid, and programmable nucleases 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 protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of one or more target nucleic acid of the plurality of target nucleic acids in the sample and wherein absence of the signal indicates an absence of the target nucleic acids in the sample. As another example, a method of assaying for a target nucleic acid in a sample, for example, comprises: a) contacting the sample to a plurality of complexes, each 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; b) contacting the plurality of complexes to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, 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. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.

[0098] 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 reporters with a detection moiety. Once the reporter is cleaved by the activated programmable nuclease, the detection moiety can be released from the reporter 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 reporter cleavage and changes upon reporter cleavage. Sometimes, the signal is absent prior to reporter cleavage and is present upon reporter cleavage. The detectable moiety generating the detectable signal can be immobilized on a support medium for detection. 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 comprises a crRNA and a trans-activating crRNA (tracrRNA).

[0099] 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 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. Guide nucleic acids, when complexed with an effector protein, may bring the effector protein into proximity of a target nucleic acid. Sufficient conditions for hybridization of a guide nucleic acid to a target nucleic acid and / or for binding of a guide nucleic acid to an effector protein include in vivo physiological conditions of a desired cell type or in vitro conditions sufficient for assaying catalytic activity of a protein, polypeptide or peptide described herein, such as the nuclease activity of an effector protein. Guide nucleic acids may comprise DNA, RNA, or a combination thereof (e.g., RNA with a thymine base). Guide nucleic acids may include a chemically modified nucleobase or phosphate backbone. Guide nucleic acids may be referred to herein as a guide RNA (gRNA). However, a guide RNA is not limited to ribonucleotides, but may comprise deoxyribonucleotides and other chemically modified nucleotides.

[0100] A guide nucleic acid may comprise a CRISPR RNA (crRNA), a short-complementarity untranslated RNA (scoutRNA), an associated trans-activating RNA (tracrRNA) or a combination thereof. The combination of a crRNA with a tracrRNA may be referred to herein as a single guide RNA (sgRNA), wherein the crRNA and the tracrRNA are covalently linked. In some embodiments, the crRNA and tracrRNA are linked by a phosphodiester bond. In some instances, the crRNA and tracrRNA are linked by one or more linked nucleotides. A guide nucleic acid may comprise a naturally occurring guide nucleic acid. A guide nucleic acid may comprise a non-naturally occurring guide nucleic acid, including a guide nucleic acid that is designed to contain a chemical or biochemical modification.

[0101] Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism. A crRNA may be the product of processing of a longer precursor CRISPR RNA (pre-crRNA) transcribed from the CRISPR array by cleavage of the pre-crRNA within each direct repeat sequence to afford shorter, mature crRNAs. A crRNA may be generated by a variety of mechanisms, including the use of dedicated endonucleases (e.g., Cas6 or Cas5d in Type I and III systems), coupling of a host endonuclease (e.g., RNase III) with tracrRNA (Type II systems), or a ribonuclease activity endogenous to the effector protein itself (e.g., Cpfl, from Type V systems). A crRNA may also be specifically generated outside of processing of a pre-crRNA and individually contacted to an effector protein in vivo or in vitro.

[0102] The CRISPR / Cas system used to detect modified target nucleic acids can comprise CRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), Cas proteins, and reporters. The term, "CRISPR RNA (crRNA)," as used herein refers to a nucleic acid comprising a first sequence, often referred to as a "spacer sequence," that hybridizes to a target sequence of a target nucleic acid, and a second sequence that either a) hybridizes to a portion of a tracrRNA or b) is capable of being non-covalently bound by an effector protein. In some embodiments, the crRNA is covalently linked to an additional nucleic acid (e.g., a tracrRNA), wherein the additional nucleic acid interacts with the effector protein.

[0103] 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 comprises 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. 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 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 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.

[0104] The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid sequence 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 sequence of a strain of coronavirus. 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 comprises 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 comprises 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 correspond to nucleic acids of a target nucleic acid; and assaying for a signal produce by cleavage of at least some reporters of a population of reporters. 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.

[0105] 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. 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.

[0106] Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of reporters with a detection moiety. Once the reporter is cleaved by the activated programmable nuclease, the detection moiety can be released from the reporter 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 reporter cleavage and changes upon reporter cleavage. Sometimes, the signal is absent prior to reporter cleavage and is present upon reporter cleavage. The detection moiety capable of generating the detectable signal can be immobilized on a support medium for detection. 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).

[0107] The devices, systems, fluidic devices, kits, and methods for detecting the presence 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 and devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, and lateral flow devices) 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 whether a target nucleic acid is present in a sample (e.g., DETECTR reactions). As disclosed herein, in some embodiments, detecting the presence or absence 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 or absence 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 or 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 reporter by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the reporter by the programmable nuclease. In other embodiments, a signal may be present in the absence of reporter 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 lateral flow device after contacting a sample with a composition comprising a programmable nuclease.

[0108] "Percent identity" and "% identity" can 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" can refer 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. Generally, computer programs can 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).

[0109] An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid when the guide nucleic acid includes a nucleotide sequence that is complementary with a target sequence in the target nucleic acid. The ability of an effector protein to detect a target nucleic acid and cleave a reporter may be dependent upon the effector protein being bound to a guide nucleic acid and the guide nucleic acid being hybridized to a target nucleic acid. An effector protein may also recognize a protospacer adjacent motif (PAM) sequence present in the target nucleic acid, which may direct the modification activity of the effector protein. An effector protein may modify a nucleic acid by cis cleavage or trans cleavage. An effector protein may be a CRISPR-associated ("Cas") protein. An effector protein may function as a single protein, including a single protein that is capable of binding to a guide nucleic acid and modifying a target nucleic acid. Alternatively, an effector protein may function as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., dimer or multimer). An effector protein, when functioning in a multiprotein complex, may have only one functional activity (e.g., binding to a guide nucleic acid), while other effector proteins present in the multiprotein complex are capable of the other functional activity (e.g., modifying a target nucleic acid). An effector protein may be a modified effector protein having reduced modification activity (e.g., a catalytically defective effector protein) or no modification activity (e.g., a catalytically inactive effector protein). Accordingly, an effector protein as used herein encompasses a modified or programmable nuclease that does not have nuclease activity.

[0110] 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 assays disclosed herein (e.g., for assaying for coronavirus in a device, such as a microfluidic device or a lateral flow assay) and methods of use thereof include a Type V or Type VI CRISPR / Cas enzyme.

[0111] The programmable nuclease system used to detect modified target nucleic acids can comprise CRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), Cas proteins, and reporters.

[0112] 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.

[0113] 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.

[0114] 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 nucleases and one or more corresponding guide nucleic acids. In some cases, the plurality of programmable nucleases are complexed with one or more corresponding guide nucleic acids. The plurality of programmable nucleases (and guide nucleic acids) can be the same. Alternatively, the plurality of programmable nuclease complexes can be different. For example, the plurality of programmable nuclease complexes can comprise different programmable nucleases and / or different guide nucleic acids associated with the programmable nucleases.

[0115] 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.

[0116] 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 to 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.

[0117] 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.

[0118] The programmable nuclease can comprise any type of human engineered enzymes. Alternatively, the programmable nuclease can comprise CRISPR enzymes derived from naturally occurring bacteria or phage. 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. 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 be a nuclease derived from a CRISPR-Cas system. The programmable nuclease can be a nuclease derived from recombineering.

[0119] 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.

[0120] 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 (also referred to as Cpf1) protein, a Cas12b protein, Cas12c protein, Cas12d protein, or a Cas12e protein.

[0121] In some embodiments, the programmable nuclease can be Cas13. Sometimes the Cas13 can be Cas13a, Cas13b, Cas13c, Cas13d, or Cas13e. In some cases, the programmable nuclease can be Mad7 or Mad2. In some cases, the programmable nuclease can be Cas12. Sometimes the Cas12 can be Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. In some cases, the Cas12 can be a Cas12 variant (SEQ ID NO: 28), which is a specific protein variant within the Cas12 protein family / classification). In some cases, the programmable nuclease can be Csm1, Cas9, C2c4, C2c8, C2c5, C2c10, C2c9, or CasZ. Sometimes, the Csm1 can also be also called smCms1, miCms1, obCms1, or suCms1. Sometimes Cas13a can also be also called C2c2. Sometimes CasZ can also be called Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. Sometimes, the programmable nuclease can be a type V CRISPR-Cas system. In some cases, the programmable nuclease can be a type VI CRISPR-Cas system. Sometimes the programmable nuclease can be a type III CRISPR-Cas system. Sometimes the programmable nuclease can be an engineered nuclease that is not from a naturally occurring CRISPR-Cas system. In some cases, the programmable nuclease can be 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.

[0122] 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 Cas 13a for trans cleavage of an RNA 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.

[0123] 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.

[0124] 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 reporters with a detection moiety. Once the reporter is cleaved by the activated programmable nuclease, the detection moiety can be released from the reporter and can generate a signal.

[0125] 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 acids 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. (Murugan et al., Mol Cell. 2017 Oct 5; 68(1): 15-25). A programmable Cas12 nuclease can be a Cas12a (also referred to as Cpf1) protein, a Cas12b protein, Cas12c protein, Cas12d protein, or a Cas12e protein. In some cases, a suitable Cas12 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to any one of SEQ ID NO: 18 - SEQ ID NO: 60. TABLE 1 - Cas12 Protein Sequences SEQ ID NO Description Sequence SEQ ID NO: 18Lachnospira ceae bacterium ND2006 (LbCas12a) SEQ ID NO: 19Acidaminoc occus sp. BV316 (AsCas12a) SEQ ID NO: 20Francisella novicida U112 (FnCas12a)SEQ ID NO: 21Porphyromo nas macacae (PmCas12a)SEQ ID NO: 22Moraxella bovoculi 237 (MbCas12a) SEQ ID NO: 23Moraxella bovoculi AAX08_00 205 (Mb2Cas12 a) SEQ ID NO: 24Moraxella bovoculi AAX11_00 205 (Mb3Cas12 a) SEQ ID NO: 25Thiomicrosp ira sp. XS5 (TsCas12a)SEQ ID NO: 26Butyrivibrio sp. NC3005 (BsCas12a)SEQ ID NO: 27AacCas12b SEQ ID NO: 28Cas 12 Variant SEQ ID NO: 29Cas 12 VariantSEQ ID NO: 30Cas 12 Variant SEQ ID NO: 31Cas 12 Variant SEQ ID NO: 32Cas 12 Variant SEQ ID NO: 33Cas 12 VariantSEQ ID NO: 34Cas 12 VariantSEQ ID NO: 35Cas 12 Variant SEQ ID NO: 36Cas 12 Variant SEQ ID NO: 37Cas 12 Variant SEQ ID NO: 38Cas 12 VariantSEQ ID NO: 39Cas 12 Variant SEQ ID NO: 40Cas 12 Variant SEQ ID NO: 41Cas 12 Variant SEQ ID NO: 42Cas 12 VariantSEQ ID NO: 43Cas 12 Variant SEQ ID NO: 44Cas 12 Variant SEQ ID NO: 45Cas 12 Variant SEQ ID NO: 46Cas 12 VariantSEQ ID NO: 47Cas 12 VariantSEQ ID NO: 48Cas 12 Variant SEQ ID NO: 49Cas 12 Variant SEQ ID NO: 50Cas 12 VariantSEQ ID NO: 51Cas 12 VariantSEQ ID NO: 52Cas 12 Variant SEQ ID NO: 53Cas 12 Variant SEQ ID NO: 54Cas 12 Variant SEQ ID NO: 55Cas 12 Variant SEQ ID NO: 56Cas 12 VariantSEQ ID NO: 57Cas 12 VariantSEQ ID NO: 58Cas 12 Variant SEQ ID NO: 59Cas 12 Variant SEQ ID NO: 60Cas 12 Variant

[0126] Alternatively, the Type V CRISPR / Cas enzyme is a programmable Cas14 nuclease. A Cas14 protein of the present disclosure includes 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 naturally occurring Cas14 protein functions as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid.

[0127] In some instances, the TypeV 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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 protein 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.

[0132] 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.

[0133] A programmable Cas14 nuclease can be a Cas14a protein, a Cas14b protein, a Cas14c protein, a Cas14d protein, a Cas14e protein, a Cas 14f protein, a Cas14g protein, a Cas14h protein, or a Cas14u protein. In some cases, a suitable Cas14 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to any one of SEQ ID NO: 61 - SEQ ID NO: 152. TABLE 2 - Cas14 Protein Sequences SEQ ID NO Sequence SEQ ID NO: 61SEQ ID NO: 62 SEQ ID NO: 63SEQ ID NO: 64SEQ ID NO: 65SEQ ID NO: 66 SEQ ID NO: 67SEQ ID NO: 68SEQ ID NO: 69SEQ ID NO: 70 SEQ ID NO: 71SEQ ID NO: 72SEQ ID NO: 73SEQ ID NO: 74SEQ ID NO: 75SEQ ID NO: 76SEQ ID NO: 77 SEQ ID NO: 78SEQ ID NO: 79SEQ ID NO: 80SEQ ID NO: 81SEQ ID NO: 82SEQ ID NO: 83 SEQ ID NO: 84SEQ ID NO: 85SEQ ID NO: 86SEQ ID NO: 87 SEQ ID NO: 88SEQ ID NO: 89SEQ ID NO: 90SEQ ID NO: 91 SEQ ID NO: 92SEQ ID NO: 93SEQ ID NO: 94SEQ IDNO: 95SEQ ID NO: 96SEQ ID NO: 97SEQ ID NO: 98SEQ ID NO: 99SEQ ID NO: 100SEQ ID NO: 101 SEQ ID NO: 102SEQ ID NO: 103SEQ ID NO: 104SEQ ID NO: 105 SEQ ID NO: 106SEQ ID NO: 107SEQ ID NO: 108SEQ ID NO: 109 SEQ ID NO: 110SEQ ID NO: 111SEQ ID NO: 112SEQ ID NO: 113 SEQ ID NO: 114SEQ ID NO: 115SEQ ID NO: 116SEQ ID NO: 117SEQ ID NO: 118SEQ ID NO: 119SEQ ID NO: 120 SEQ ID NO: 121SEQ ID NO: 122SEQ ID NO: 123SEQ ID NO: 124SEQ ID NO: 125SEQ ID NO: 126 SEQ ID NO: 127SEQ ID NO: 128SEQ ID NO: 129SEQ ID NO: 130SEQ ID NO: 131SEQ ID NO: 132SEQ ID NO: 133SEQ ID NO: 134SEQ ID NO: 135SEQ ID NO: 136SEQ ID NO: 137SEQ ID NO: 138SEQ ID NO: 139SEQ ID NO: 140SEQ ID NO: 141SEQ ID NO: 142SEQ ID NO: 143SEQ ID NO: 144 SEQ ID NO: 145SEQ ID NO: 146SEQ ID NO: 147SEQ ID NO: 148SEQ ID NO: 149SEQ ID NO: 150SEQ ID NO: 151SEQ ID NO: 152

[0134] In some embodiments, the Type V CRISPR / Cas enzyme is a CasΦ nuclease. A CasΦ polypeptide can function as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid. A programmable CasΦ nuclease of the present disclosure 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 CasΦ nuclease especially advantageous for genome engineering and new functionalities for genome manipulation.

[0135] TABLE 3 provides amino acid sequences of illustrative CasΦ polypeptides that can be used in compositions and methods of the disclosure. TABLE 3 - CasΦ Amino Acid Sequences Name SEQ ID NO Amino Acid Sequence CasΦ.1SEQ ID NO: 221CasΦ.2SEQ ID NO: 222 CasΦ.3SEQ ID NO: 223 CasΦ.4SEQ ID NO: 224CasΦ.5SEQ ID NO: 225 CasΦ.6SEQ ID NO: 226CasΦ.7SEQ ID NO: 227 CasΦ.8SEQ ID NO: 228CasΦ.9SEQ ID NO: 229 CasΦ.10SEQ ID NO: 230CasΦ.11SEQ ID NO: 231 CasΦ.12SEQ ID NO: 232 CasΦ.13SEQ ID NO: 233CasΦ.14SEQ ID NO: 234 CasΦ.15SEQ ID NO: 235CasΦ.16SEQ ID NO: 236 CasΦ.17SEQ ID NO: 237CasΦ.18SEQ ID NO: 238 CasΦ.19SEQ ID NO: 239 CasΦ.20SEQ ID NO: 240CasΦ.21SEQ ID NO: 241 CasΦ.22SEQ ID NO: 242CasΦ.23SEQ ID NO: 243 CasΦ.24SEQ ID NO: 244CasΦ.25SEQ ID NO: 245CasΦ.26SEQ ID NO: 246 CasΦ.27SEQ ID NO: 247CasΦ.28SEQ ID NO: 248 CasΦ.29SEQ ID NO: 249CasΦ.30SEQ ID NO: 250 CasΦ.31SEQ ID NO: 251CasΦ.32SEQ ID NO: 252 CasΦ.33SEQ ID NO: 253CasΦ.41SEQ ID NO: 254 CasΦ.34SEQ ID NO: 255CasΦ.35SEQ ID NO: 256 CasΦ.43SEQ ID NO: 257 CasΦ.44SEQ ID NO: 258CasΦ.36SEQ ID NO: 259 CasΦ.37SEQ ID NO: 260CasΦ.45SEQ ID NO: 261 CasΦ.38SEQ ID NO: 262CasΦ.39SEQ ID NO: 263 CasΦ.42SEQ ID NO: 264CasΦ.46SEQ ID NO: 265 CasΦ.47SEQ ID NO: 266CasΦ.48SEQ ID NO: 267CasΦ.49SEQ ID NO: 268 (Bold sequence is Nuclear Localization Signal)

[0136] In some embodiments, any of the programmable CasΦ nuclease of the present disclosure (e.g., any one of SEQ ID NO: 221 - SEQ ID NO: 268 or fragments or variants thereof) may include a nuclear localization signal (NLS). In some cases, said NLS may have a sequence of KRPAATKKAGQAKKKKEF (SEQ ID NO: 269).

[0137] A CasΦ polypeptide or a variant thereof can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 221 - SEQ ID NO: 268.

[0138] In some embodiments, the Type VI CRISPR / Cas enzyme 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 (Liu et al., Cell 2017 Jan 12;168(l-2):121-134.el2). The HEPN domains each comprise aR-X 4 -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. (Tambe et al., Cell Rep. 2018 Jul 24; 24(4): 1025-1036.). 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

[0139] A programmable Cas13 nuclease can be a Cas13a protein (also referred to as "c2c2"), a Cas13b protein, a Cas13c protein, a Cas13d protein, or a Cas13e protein. Example C2c2 proteins are set forth as SEQ ID NO: 153 - SEQ ID NO: 160. In some cases, a subject C2c2 protein includes an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NO: 153 - SEQ ID NO: 160. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Listeria seeligeri C2c2 amino acid sequence set forth in SEQ ID NO: 153. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Leptotrichia buccalis C2c2 amino acid sequence set forth in SEQ ID NO: 154. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Rhodobacter capsulatus C2c2 amino acid sequence set forth in SEQ ID NO: 156. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Carnobacterium gallinarum C2c2 amino acid sequence set forth in SEQ ID NO: 157. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Herbinix hemicellulosilytica C2c2 amino acid sequence set forth in SEQ ID NO: 158. In some cases, the C2c2 protein includes an amino acid sequence having 80% or more amino acid sequence identity with the Leptotrichia buccalis (Lbu) C2c2 amino acid sequence set forth in SEQ ID NO: 154. In some cases, the C2c2 protein is a Leptotrichia buccalis (Lbu) C2c2 protein (e.g., see SEQ ID NO: 154). In some cases, the C2c2 protein includes the amino acid sequence set forth in any one of SEQ ID NO: 153, SEQ ID NO: 154 and SEQ ID NO: 156 - SEQ ID NO: 160. In some cases, a C2c2 protein used in a method of the present disclosure is not a Leptotrichia shahii (Lsh) C2c2 protein. In some cases, a C2c2 protein used in a method of the present disclosure is not a C2c2 polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Lsh C2c2 polypeptide set forth in SEQ ID NO: 155. Other Cas13 protein sequences are set forth in SEQ ID NO: 153 - SEQ ID NO: 170. TABLE 4 - Cas13 Protein Sequences SEQ ID NO Description Sequence SEQ ID NO: 153Listeria seeligeri C2c2 amino acid sequenceSEQ ID NO: 154Leptotrichia buccalis (Lbu) C2c2 amino acid sequence SEQ ID NO: 155Leptotrichia shahii (Lsh) C2c2 protein SEQ ID NO: 156Rhodobacter capsulatus C2c2 amino acid sequenceSEQ ID NO: 157Camobacter ium gallinarum C2c2 amino acid sequenceSEQ ID NO: 158Herbinix hemicellulos ilytica C2c2 amino acid sequence SEQ ID NO: 159Paludibacter propionicige nes C2c2 amino acid sequence SEQ ID NO: 160Leptotrichia wadei (Lwa) C2c2 amino acid sequenceSEQ ID NO: 161Bergeyella zoohelcum Cas 13b SEQ ID NO: 162Prevotella intermedia Cas 13b SEQ ID NO: 163Prevotella buccae Cas 13bSEQ ID NO: 164Porphyromo nas gingivalis Cas 13b SEQ ID NO: 165Bacteroides pyogenes Cas13b SEQ ID NO: 166Cas13cSEQ ID NO: 167Cas13cSEQ ID NO: 168Cas13cSEQ ID NO: 169Cas13c SEQ ID NO: 170Cas13c

[0140] In some cases, a suitable programmable nuclease for use in the compositions and methods herein comprises an amino acid sequence having at least 60% amino acid sequence identity to any one of SEQ ID NO: 396 - SEQ ID NO: 423. In some cases, a suitable programmable nuclease for use in the compositions and methods herein comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NO: 396 - SEQ ID NO: 423. In some cases, a subject programmable nuclease for use in the compositions and methods herein may include an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) amino acid sequence identity with the amino acid seauence set forth in any one of SEQ ID NO: 396 - SEQ ID NO: 423. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 396. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 397. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 398. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 399. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 400. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 401. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 402. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 403. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 404. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 405. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 406. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 407. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 408. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 409. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 410. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 411. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 412. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 413. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 414. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 415. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 416. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 417. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 418. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 419. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 420. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 421. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 422. In some cases, a suitable programmable nuclease polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 423. Table 20: Amino Acid Sequences of Exemplary Programmable NucleasesSEQ ID NO: Programmable Nuclease Amino Acid Sequence SEQ ID NO: 396 SEQ ID NO: 397 SEQ ID NO: 398 SEQ ID NO: 399 SEQ ID NO: 400 SEQ ID NO: 401 SEQ ID NO: 402 SEQ ID NO: 403 SEQ ID NO: 404 SEQ ID NO: 405 SEQ ID NO: 406 SEQ ID NO: 407 SEQ ID NO: 408 SEQ ID NO: 409 SEQ ID NO: 410 SEQ ID NO: 411 SEQ ID NO: 412 SEQ ID NO: 413 SEQ ID NO: 414 SEQ ID NO: 415 SEQ ID NO: 416 SEQ ID NO: 417 SEQ ID NO: 418 SEQ ID NO: 419 SEQ ID NO: 420 SEQ ID NO: 421 SEQ ID NO: 422 SEQ ID NO: 423

[0141] The programmable nuclease can be Cas13. Sometimes the Cas13 can be Cas13a, Cas13b, Cas13c, Cas13d, or Cas13e. In some cases, the programmable nuclease can be Mad7 or Mad2. In some cases, the programmable nuclease can be Cas12. Sometimes the Cas12 can be Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. In some cases, the programmable nuclease can be Csm1, Cas9, C2c4, C2c8, C2c5, C2c10, C2c9, or CasZ. Sometimes, the Csm1 can also be also called smCms1, miCms1, obCms1, or suCms1. Sometimes Cas13a can also be also called C2c2. Sometimes CasZ can also be called Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14i, Cas14j, or Cas14k. Sometimes, the programmable nuclease can be a type V CRISPR-Cas system. In some cases, the programmable nuclease can be a type VI CRISPR-Cas system. Sometimes the programmable nuclease can be a type III CRISPR-Cas system. In some cases, the programmable nuclease can be 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.

[0142] 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., Cas13). 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 RNA reporter. An RNA reporter can be an RNA-based reporter molecule. 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., Cas13, 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 may 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 may 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 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. 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.Engineered programmable nucleases

[0143] 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, 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.

[0144] 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.

[0145] 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 the 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.

[0146] 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.Thermostable programmable nuclease

[0147] 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 55% 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 60 % 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 65% 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 70 % 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 75% 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 80% 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 85% 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 90% 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 95% 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 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 2-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 3-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 4-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 5-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 6-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 7-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 8-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 9-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 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, at least 50-fold or more of that at 37 °C.

[0148] 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 55 % 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 60 % 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 65 % 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 70 % 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 75 % 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 80 % 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 85 % 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 90 % 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 95 % 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 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 2-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 3-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 4-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 5-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 6-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 7-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 8-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 9-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 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.

[0149] 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 55 % 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 60 % 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 65 % 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 70 % 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 75 % 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 80 % 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 85 % 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 90 % 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 95 % 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 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 2-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 3-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 4-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 5-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 6-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 7-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 8-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 9-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 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.

[0150] 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 55 % 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 60 % 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 65 % 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 70 % 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 75 % 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 80 % 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 85 % 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 90 % 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 95 % 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 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 2-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 3-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 4-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 5-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 6-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 7-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 8-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 9-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 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.

[0151] 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 55 % 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 60 % 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 65 % 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 70 % 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 75 % 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 80 % 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 85 % 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 90 % 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 95 % 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 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 2-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 3-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 4-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 5-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 6-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 7-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 8-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 9-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 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-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.

[0152] 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 55 % 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 60 % 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 65 % 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 70 % 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 75 % 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 80 % 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 85 % 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 90 % 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 95 % 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 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 2-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 3-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 4-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 5-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 6-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 7-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 8-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 9-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 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. In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 70 °C, 75 °C. 80 °C, or more may be at least 50, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, 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 , at least 10-fold , 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.

[0153] 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-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 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 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.Reporters

[0154] Described herein are reagents comprising a reporter comprising a detection moiety, wherein the reporter is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal. As used herein, a reporter is used interchangeably with reporter molecule. In some cases, the reporter comprises a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the reporter comprises a single-stranded nucleic acid comprising ribonucleotides. The reporter can comprise a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the reporter comprises 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 reporter comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the reporter 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 reporter 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 reporter has only ribonucleotide residues. In some cases, the reporter has only deoxyribonucleotide residues. In some cases, the reporter comprises nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the reporter comprises synthetic nucleotides. In some cases, the reporter comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the reporter is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length. In some cases, the reporter comprises at least one uracil ribonucleotide. In some cases, the reporter comprises at least two uracil ribonucleotides. Sometimes the reporter has only uracil ribonucleotides. In some cases, the reporter comprises at least one adenine ribonucleotide. In some cases, the reporter comprises at least two adenine ribonucleotide. In some cases, the reporter has only adenine ribonucleotides. In some cases, the reporter comprises at least one cytosine ribonucleotide. In some cases, the reporter comprises at least two cytosine ribonucleotide. In some cases, the reporter comprises at least one guanine ribonucleotide. In some cases, the reporter comprises at least two guanine ribonucleotide. A reporter can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the reporter is from 5 to12 nucleotides in length. In some cases, the reporter 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 reporter 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 reporter can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Cas12, a reporter can be 10 nucleotides in length. A DETECTR reaction can comprise 100 pM to 1 nM of reporters. A DETECTR reaction can comprise 1 nM to 5 nM of reporters. A DETECTR reaction can comprise 5 nM to 20 nM of reporters. A DETECTR reaction can comprise 20 nM to 50 nM of reporters. A DETECTR reaction can comprise 50 nM to 100 nM of reporters. A DETECTR reaction can comprise 100 nM to 250 nM reporters. A DETECTR reaction can comprise 250 nM to 500 nM reporters. A DETECTR reaction can comprise 500 nM to 1000 nM (1 µM) of reporters.

[0155] The reporter can comprise a detection moiety (in addition to the nucleic acid) capable of generating a first detectable signal. Sometimes the reporter 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-electric 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 reporter, 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 reporters. Sometimes, a calorimetric signal is heat absorbed after cleavage of the reporters. A potentiometric signal, for example, is electrical potential produced after cleavage of the reporters. An amperometric signal can be movement of electrons produced after the cleavage of a reporter. 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 reporters. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of reporters. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the reporter.

[0156] 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 reporter by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the reporter by the programmable nuclease. In other embodiments, a signal can be present in the absence of reporter 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 or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.

[0157] In some cases, the signal can comprise a colorimetric signal or a signal visible by eye. In some instances, the signal is fluorescent, electrical, chemical, electrochemical, or magnetic. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, the detectable signal is a colorimetric signal or a signal visible by eye. In some instances, the detectable signal is fluorescent, electrical, chemical, electrochemical, or magnetic. In some cases, the first detection signal is generated by binding of the detection moiety to the capture molecule in the detection region, where the first detection signal indicates that the sample contained the target nucleic acid. Sometimes the system can detect more than one type of target nucleic acid, wherein the system may comprise more than one type of guide nucleic acid and more than one type of reporter. In some cases, the detectable signal is generated directly by the cleavage event. Alternatively, or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal is a colorimetric or color-based signal. In some cases, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium.

[0158] The reporter can comprise a quenching moiety 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 nucleic acid of the reporter. Sometimes the detection moiety is at the 3' terminus of the nucleic acid of the reporter. In some cases, the detection moiety is at the 5' terminus of the nucleic acid of the reporter. In some cases, the quenching moiety is at the 3' terminus of the nucleic acid of the reporter. In some cases, the reporter comprises at least one population of the single-stranded nucleic acid capable of generating a first detectable signal. In some cases, the reporter comprises a population of the single-stranded nucleic acid capable of generating a first detectable signal. Optionally, there is more than one population of reporter. 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 reporters capable of generating a detectable signal.

[0159] In some cases, the one or more detectable signals generated after cleavage can produce an index of refraction change or one or more electrochemical changes. In some cases, real-time detection of the Cas reaction can be achieved using fluorescence, electrochemical detection, and / or electrochemiluminescence. 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 comprise a digital camera, such as 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 device 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).

[0160] In certain aspects of this disclosure, multiplexing refers to parallel sensing of multiple target nucleic acid sequences in one sample by multiple probes. 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 reporters 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 reporters capable of generating a detectable signal. TABLE 5 - Exemplary Single Stranded Reporter 5' DETECTION MOIETY* SEQUENCE (SEQ ID NO:) 3' QUENCHER* / 56-FAM / rUrUrUrUrU (SEQ ID NO: 1) / 3IABkFQ / / 5IRD700 / rUrUrUrUrU (SEQ ID NO: 1) / 3IRQC1N / / 5TYE665 / rUrUrUrUrU (SEQ ID NO: 1) / 3IAbRQSp / / 5Alex594N / rUrUrUrUrU (SEQ ID NO: 1) / 3IAbRQSp / / 5ATTO633N / rUrUrUrUrU (SEQ ID NO: 1) / 3IAbRQSp / / 56-FAM / rUrUrUrUrUrUrUrU(SEQ ID NO : 2) / 3IABkFQ / / 5IRD700 / rUrUrUrUrUrUrUrU(SEQ ID NO : 2) / 3IRQC1N / / 5TYE665 / rUrUrUrUrUrUrUrU(SEQ ID NO : 2) / 3IAbRQSp / / 5Alex594N / rUrUrUrUrUrUrUrU(SEQ ID NO : 2) / 3IAbRQSp / / 5ATTO633N / rUrUrUrUrUrUrUrU(SEQ ID NO : 2) / 3IAbRQSp / / 56-FAM / rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 3) / 3IABkFQ / / 5IRD700 / rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 3) / 3IRQC1N / / 5TYE665 / rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 3) / 3IAbRQSp / / 5Alex594N / rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 3) / 3IAbRQSp / / 5ATTO633N / rUrUrUrUrUrUrUrUrUrU(SEQ ID NO: 3) / 3IAbRQSp / / 56-FAM / TTTTrUrUTTTT(SEQ ID NO: 4) / 3IABkFQ / / 5IRD700 / TTTTrUrUTTTT(SEQ ID NO : 4) / 3IRQC1N / / 5TYE665 / TTTTrUrUTTTT(SEQ ID NO : 4) / 3IAbRQSp / / 5Alex594N / TTTTrUrUTTTT(SEQ ID NO : 4) / 3IAbRQSp / / 5ATTO633N / TTTTrUrUTTTT(SEQ ID NO : 4) / 3IAbRQSp / / 56-FAM / TTrUrUTT(SEQ ID NO: 5) / 3IABkFQ / / 5IRD700 / TTrUrUTT(SEQ ID NO: 5) / 3IRQC1N / / 5TYE665 / TTrUrUTT(SEQ ID NO: 5) / 3IAbRQSp / / 5Alex594N / TTrUrUTT(SEQ ID NO: 5) / 3IAbRQSp / / 5ATTO633N / TTrUrUTT(SEQ ID NO: 5) / 3IAbRQSp / / 56-FAM / TArArUGC(SEQ ID NO: 6) / 3IABkFQ / / 5IRD700 / TArArUGC(SEQ ID NO : 6) / 3IRQC1N / / 5TYE665 / TArArUGC(SEQ ID NO: 6) / 3IAbRQSp / / 5Alex594N / TArArUGC(SEQ ID NO: 6) / 3IAbRQSp / / 5ATTO633N / TArArUGC(SEQ ID NO: 6) / 3IAbRQSp / / 56-FAM / TArUrGGC(SEQ ID NO: 7) / 3IABkFQ / / 5IRD700 / TArUrGGC(SEQ ID NO: 7) / 3IRQC1N / / 5TYE665 / TArUrGGC(SEQ ID NO: 7) / 3IAbRQSp / / 5Alex594N / TArUrGGC(SEQ ID NO: 7) / 3IAbRQSp / / 5ATTO633N / TArUrGGC(SEQ ID NO: 7) / 3IAbRQSp / / 56-FAM / rUrUrUrUrU(SEQ ID NO: 8) / 3IABkFQ / / 5IRD700 / rUrUrUrUrU(SEQ ID NO: 8) / 3IRQC1N / / 5TYE665 / rUrUrUrUrU(SEQ ID NO: 8) / 3IAbRQSp / / 5Alex594N / rUrUrUrUrU(SEQ ID NO: 8) / 3IAbRQSp / / 5ATTO633N / rUrUrUrUrU(SEQ ID NO: 8) / 3IAbRQSp / / 56-FAM / TTATTATT (SEQ ID NO: 9) / 3IABkFQ / / 56-FAM / TTATTATT (SEQ ID NO: 9) / 3IABkFQ / / 5IRD700 / TTATTATT (SEQ ID NO: 9) / 3IRQC1N / / 5TYE665 / TTATTATT (SEQ ID NO: 9) / 3IAbRQSp / / 5Alex594N / TTATTATT (SEQ ID NO: 9) / 3IAbRQSp / / 5ATTO633N / TTATTATT (SEQ ID NO: 9) / 3IAbRQSp / / 56-FAM / TTTTTT (SEQ ID NO: 10) / 3IABkFQ / / 56-FAM / TTTTTTTT (SEQ ID NO: 11) / 3IABkFQ / / 56-FAM / TTTTTTTTTT (SEQ ID NO: 12) / 3IABkFQ / / 56-FAM / TTTTTTTTTTTT (SEQ ID NO: 13) / 3IABkFQ / / 56-FAM / TTTTTTTTTTTTTT (SEQ ID NO: 14) / 3IABkFQ / / 56-FAM / AAAAAA (SEQ ID NO: 15) / 3IABkFQ / / 56-FAM / CCCCCC (SEQ ID NO: 16) / 3IABkFQ / / 56-FAM / GGGGGG (SEQ ID NO: 17) / 3IABkFQ / / 56-FAM / TTATTATT (SEQ ID NO: 9) / 3IABkFQ / / 56-FAM / : 5' 6-Fluorescein (Integrated DNA Technologies) / 3IABkFQ / : 3' Iowa Black FQ (Integrated DNA Technologies) / 5IRD700 / : 5' IRDye 700 (Integrated DNA Technologies) / 5TYE665 / : 5' TYE 665 (Integrated DNA Technologies) / 5Alex594N / : 5' Alexa Fluor 594 (NHS Ester) (Integrated DNA Technologies) / 5TTO633N / : 5' ATTO TM 633 (NHS Ester) (Integrated DNA Technologies) / 3IRQC1N / : 3' IRDye QC-1 Quencher (Li-Cor) / 3IAbRQSp / : 3' Iowa Black RQ (Integrated DNA Technologies) rU: uracil ribonucleotide rG: guanine ribonucleotide *This Table refers to the detection moiety and quencher moiety as their tradenames and their source is identified. However, alternatives, generics, or non-tradename moieties with similar function from other sources can also be used.

[0161] A detection moiety can be an infrared fluorophore. A detection moiety can be a fluorophore that emits fluorescence in the range of from 500 nm and 720 nm. A detection moiety can be a fluorophore that emits fluorescence in the range of from 500 nm and 720 nm. In some cases, the detection moiety emits fluorescence at a wavelength of 700 nm or higher. In other cases, the detection moiety emits fluorescence at about 660 nm or about 670 nm. In some cases, the detection moiety emits fluorescence at in the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 6890 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. A detection moiety can be a fluorophore that emits a fluorescence in the same range as 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor, or ATTO TM 633 (NHS Ester). A detection moiety can be fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). A detection moiety can be a fluorophore that emits a fluorescence in the same range as 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). A detection moiety can be fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). Any of the detection moieties described herein can be from any commercially available source, can be an alternative with a similar function, a generic, or a non-tradename of the detection moieties listed.

[0162] A detection moiety can be chosen for use based on the type of sample to be tested. For example, a detection moiety that is an infrared fluorophore is used with a urine sample. As another example, SEQ ID NO: 1 with a fluorophore that emits around 520 nm is used for testing in non-urine samples, and SEQ ID NO: 8 with a fluorophore that emits a fluorescence around 700 nm is used for testing in urine samples.

[0163] A quenching moiety can be chosen based on its ability to quench the detection moiety. A quenching moiety can be a non-fluorescent fluorescence quencher. A quenching moiety can quench a detection moiety that emits fluorescence in the range of from 500 nm and 720 nm. A quenching moiety can quench a detection moiety that emits fluorescence in the range of from 500 nm and 720 nm. In some cases, the quenching moiety quenches a detection moiety that emits fluorescence at a wavelength of 700 nm or higher. In other cases, the quenching moiety quenches a detection moiety that emits fluorescence at about 660 nm or about 670 nm. In some cases, the quenching moiety quenches a detection moiety emits fluorescence at in the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 6890 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. A quenching moiety can quench fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). A quenching moiety can be Iowa Black RQ, Iowa Black FQ or IRDye QC-1 Quencher. A quenching moiety can quench fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). A quenching moiety can be Iowa Black RQ (Integrated DNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDye QC-1 Quencher (LiCor). Any of the quenching moieties described herein can be from any commercially available source, can be an alternative with a similar function, a generic, or a non-tradename of the quenching moieties listed.

[0164] 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. In some cases, the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some cases, the detection moiety comprises an infrared (IR) dye. In some cases, the detection moiety comprises an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety comprises a polypeptide. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some instances, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some instances, the detection moiety comprises a gold nanoparticle or a latex nanoparticle.

[0165] Alternatively, or in combination, in some embodiments, detecting the presence or absence 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 or 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 reporter by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the reporter by the programmable nuclease. In other embodiments, a signal may be present in the absence of reporter 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 lateral flow device after contacting a sample with a composition comprising a programmable nuclease.

[0166] A detection moiety can be any moiety capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. A reporter, sometimes, is protein-nucleic acid that is capable of generating 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 reporters. Sometimes, a calorimetric signal is heat absorbed after cleavage of the reporters. A potentiometric signal, for example, is electrical potential produced after cleavage of the reporters. An amperometric signal can be movement of electrons produced after the cleavage of a reporter. 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 reporters. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of reporters. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the reporter.

[0167] Often, the protein-nucleic acid is an enzyme-nucleic acid. The enzyme may be sterically hindered when present as in the enzyme-nucleic acid, but then functional upon cleavage from the nucleic acid. Often, the enzyme is an enzyme that produces a reaction with a substrate. An enzyme can be invertase. Often, the substrate of invertase is sucrose and DNS reagent.

[0168] Sometimes the protein-nucleic acid is a substrate-nucleic acid. Often the substrate is a substrate that produces a reaction with an enzyme.

[0169] A protein-nucleic acid may be attached to a solid support. The solid support, for example, may be a surface. A surface can be an electrode. Sometimes the solid support is a bead. Often the bead is a magnetic bead. Upon cleavage, the protein is liberated from the solid and interacts with other mixtures. For example, the protein is an enzyme, and upon cleavage of the nucleic acid of the enzyme-nucleic acid, the enzyme flows through a chamber into a mixture comprising the substrate. When the enzyme meets the enzyme substrate, a reaction occurs, such as a colorimetric reaction, which is then detected. As another example, the protein is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme flows through a chamber into a mixture comprising the enzyme. When the enzyme substrate meets the enzyme, a reaction occurs, such as a calorimetric reaction, which is then detected.

[0170] In some embodiments, the reporter comprises a nucleic acid conjugated to an affinity molecule and the affinity molecule conjugated to the fluorophore (e.g., nucleic acid - affinity molecule - fluorophore) or the nucleic acid conjugated to the fluorophore and the fluorophore conjugated to the affinity molecule (e.g., nucleic acid - fluorophore - affinity molecule). In some embodiments, a linker conjugates the nucleic acid to the affinity molecule. In some embodiments, a linker conjugates the affinity molecule to the fluorophore. In some embodiments, a linker conjugates the nucleic acid to the fluorophore. A linker can be any suitable linker known in the art. In some embodiments, the nucleic acid of the reporter can be directly conjugated to the affinity molecule and the affinity molecule can be directly conjugated to the fluorophore or the nucleic acid can be directly conjugated to the fluorophore and the fluorophore can be directly conjugated to the affinity molecule. In this context, "directly conjugated" indicated that no intervening molecules, polypeptides, proteins, or other moieties are present between the two moieties directly conjugated to each other. For example, if a reporter comprises a nucleic acid directly conjugated to an affinity molecule and an affinity molecule directly conjugated to a fluorophore - no intervening moiety is present between the nucleic acid and the affinity molecule and no intervening moiety is present between the affinity molecule and the fluorophore. The affinity molecule can be biotin, avidin, streptavidin, or any similar molecule.

[0171] In some cases, the reporter comprises a substrate-nucleic acid. The substrate may be sequestered from its cognate enzyme when present as in the substrate-nucleic acid, but then is released from the nucleic acid upon cleavage, wherein the released substrate can contact the cognate enzyme to produce a detectable signal. Often, the substrate is sucrose and the cognate enzyme is invertase, and a DNS reagent can be used to monitor invertase activity.

[0172] A major advantage of the devices and methods disclosed herein is the design of excess reporters to total nucleic acids in an unamplified or an amplified sample, not including the nucleic acid of the reporter. Total nucleic acids can include the target nucleic acids and non-target nucleic acids, not including the nucleic acid of the reporter. The non-target nucleic acids can be from the original sample, either lysed or unlysed. The non-target nucleic acids can also be byproducts of amplification. Thus, the non-target nucleic acids can include both non-target nucleic acids from the original sample, lysed or unlysed, and from an amplified sample. The presence of a large amount of non-target nucleic acids, an activated programmable nuclease may be inhibited in its ability to bind and cleave the reporter sequences. This is because the activated programmable nucleases collaterally cleaves any nucleic acids. If total nucleic acids are present in large amounts, they may outcompete reporters for the programmable nucleases. The devices and methods disclosed herein are designed to have an excess of reporter to total nucleic acids, such that the detectable signals from cleavage reactions (e.g., DETECTR reactions) are particularly superior. In some embodiments, the reporter can be present in at least 1.5 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, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, from 1.5 fold to 100 fold, from 2 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 60 fold, from 60 fold to 70 fold, from 70 fold to 80 fold, from 80 fold to 90 fold, from 90 fold to 100 fold, from 1.5 fold to 10 fold, from 1.5 fold to 20 fold, from 10 fold to 40 fold, from 20 fold to 60 fold, or from 10 fold to 80 fold excess of total nucleic acids.

[0173] A second significant advantage of the devices and methods disclosed herein is the design of an excess volume comprising the guide nucleic acid, the programmable nuclease, and the reporter, which contacts a smaller volume comprising the sample with the target nucleic acid of interest. The smaller volume comprising the sample can be unlysed sample, lysed sample, or lysed sample which has undergone any combination of reverse transcription, amplification, and in vitro transcription, as claimed. The presence of various reagents in a crude, non-lysed sample, a lysed sample, or a lysed and amplified sample, such as buffer, magnesium sulfate, salts, the pH, a reducing agent, primers, dNTPs, NTPs, cellular lysates, non-target nucleic acids, primers, or other components, can inhibit the ability of the programmable nuclease to find and cleave the nucleic acid of the reporter. This may be due to nucleic acids that are not the reporter, which outcompete the nucleic acid of the reporter, for the programmable nuclease. Alternatively, various reagents in the sample may simply inhibit the activity of the programmable nuclease. Thus, the devices and methods provided herein for contacting an excess volume comprising the guide nucleic acid, the programmable nuclease, and the reporter to a smaller volume comprising the sample with the target nucleic acid of interest provides for superior detection of the target nucleic acid by ensuring that the programmable nuclease is able to find and cleaves the nucleic acid of the reporter. In some embodiments, the volume comprising the guide nucleic acid, the programmable nuclease, and the reporter (can be referred to as "a second volume") is 4-fold greater than a volume comprising the sample (can be referred to as "a first volume"). In some embodiments, the volume comprising the guide nucleic acid, the programmable nuclease, and the reporter (can be referred to as "a second volume") is at least 1.5 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, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, from 1.5 fold to 100 fold, from 2 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 60 fold, from 60 fold to 70 fold, from 70 fold to 80 fold, from 80 fold to 90 fold, from 90 fold to 100 fold, from 1.5 fold to 10 fold, from 1.5 fold to 20 fold, from 10 fold to 40 fold, from 20 fold to 60 fold, or from 10 fold to 80 fold greater than a volume comprising the sample (can be referred to as "a first volume"). In some embodiments, the volume comprising the sample is at least 0.5 ul, at least 1 ul, at least at least 1 µL, at least 2 µL, at least 3 µL, at least 4 µL, at least 5 µL, at least 6 µL, at least 7 µL, at least 8 µL, at least 9 µL, at least 10 µL, at least 11 µL, at least 12 µL, at least 13 µL, at least 14 µL, at least 15 µL, at least 16 µL, at least 17 µL, at least 18 µL, at least 19 µL, at least 20 µL, at least 25 µL, at least 30 µL, at least 35 µL, at least 40 µL, at least 45 µL, at least 50 µL, at least 55 µL, at least 60 µL, at least 65 µL, at least 70 µL, at least 75 µL, at least 80 µL, at least 85 µL, at least 90 µL, at least 95 µL, at least 100 µL, from 0.5 µL to 5 ul µL, from 5 µL to 10 µL, from 10 µL to 15 µL, from 15 µL to 20 µL, from 20 µL to 25 µL, from 25 µL to 30 µL, from 30 µL to 35 µL, from 35 µL to 40 µL, from 40 µL to 45 µL, from 45 µL to 50 µL, from 10 µL to 20 µL, from 5 µL to 20 µL, from 1 µL to 40 µL, from 2 µL to 10 µL, or from 1 µL to 10 µL. In some embodiments, the volume comprising the programmable nuclease, the guide nucleic acid, and the reporter is at least 10 µL, at least 11 µL, at least 12 µL, at least 13 µL, at least 14 µL, at least 15 µL, at least 16 µL, at least 17 µL, at least 18 µL, at least 19 µL, at least 20 µL, at least 21 µL, at least 22 µL, at least 23 µL, at least 24 µL, at least 25 µL, at least 26 µL, at least 27 µL, at least 28 µL, at least 29 µL, at least 30 µL, at least 40 µL, at least 50 µL, at least 60 µL, at least 70 µL, at least 80 µL, at least 90 µL, at least 100 µL, at least 150 µL, at least 200 µL, at least 250 µL, at least 300 µL, at least 350 µL, at least 400 µL, at least 450 µL, at least 500 µL, from 10 µL to 15 ul µL, from 15 µL to 20 µL, from 20 µL to 25 µL, from 25 µL to 30 µL, from 30 µL to 35 µL, from 35 µL to 40 µL, from 40 µL to 45 µL, from 45 µL to 50 µL, from 50 µL to 55 µL, from 55 µL to 60 µL, from 60 µL to 65 µL, from 65 µL to 70 µL, from 70 µL to 75 µL, from 75 µL to 80 µL, from 80 µL to 85 µL, from 85 µL to 90 µL, from 90 µL to 95 µL, from 95 µL to 100 µL, from 100 µL to 150 µL, from 150 µL to 200 µL, from 200 µL to 250 µL, from 250 µL to 300 µL, from 300 µL to 350 µL, from 350 µL to 400 µL, from 400 µL to 450 µL, from 450 µL to 500 µL, from 10 µL to 20 µL, from 10 µL to 30 µL, from 25 µL to 35 µL, from 10 µL to 40 µL, from 20 µL to 50 µL, from 18 µL to 28 µL, or from 17 µL to 22 µL.

[0174] A reporter may be a hybrid nucleic acid reporter. A hybrid nucleic acid reporter comprises a nucleic acid with at least one deoxyribonucleotide and at least one ribonucleotide. In some embodiments, the nucleic acid of the hybrid nucleic acid reporter can be of any length and can have any mixture of DNAs and RNAs. For example, in some cases, longer stretches of DNA can be interrupted by a few ribonucleotides. Alternatively, longer stretches of RNA can be interrupted by a few deoxyribonucleotides. Alternatively, every other base in the nucleic acid may alternate between ribonucleotides and deoxyribonucleotides. A major advantage of the hybrid nucleic acid reporter is increased stability as compared to a pure RNA nucleic acid reporter. For example, a hybrid nucleic acid reporter can be more stable in solution, lyophilized, or vitrified as compared to a pure DNA or pure RNA reporter.

[0175] Additionally, target nucleic acid can be amplified before binding to the crRNA of the CRISPR enzyme. This amplification can be PCR amplification or isothermal amplification. This nucleic acid amplification of the sample can improve at least one of sensitivity, specificity, or accuracy of the detection the target RNA. 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, 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 reaction is performed at a temperature of around 20-45°C. The nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C. The nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, or 45°C.

[0176] In some cases, an amplification method may comprise a set of multiple distinct primers targeting a single amplicon (e.g., a gene, such as the SARS-Cov-2 E and N genes). The set of primers may all target sequences upstream of a gene of interest, and may comprise overlapping, partially overlapping, or entirely distinct sequences. The use of multiple primers can increase the yield from amplification reactions. In some cases, the use of multiple distinct primers can increase the likelihood that damaged nucleic acids are amplified in a reaction. The use of multiple distinct primers can also facilitate amplification (and therefore detection) of mutant nucleic acids. In some cases, a sample may comprise a mutant strain of SARS-CoV-2 that would not be detectable if a single primer were used for amplification. A primer may be designed for thermal cycling amplification of the target nucleic acid. A primer may be designed for isothermal amplification of the target nucleic acid.

[0177] In some embodiments, an amplification method may utilize an amplification activator. A set of amplification reagents may require addition of an amplification activator for activity. In such cases, an amplification activator may enable temporal control over the start of an amplification reaction. In some embodiments, the amplification activator comprises a magnesium salt, deoxyribonucleoside triphosphates (dNTPs), nucleoside triphosphates (NTPs), adenosine triphosphate (ATP), or a combination thereof. In some embodiments, the magnesium salt is magnesium sulfate, magnesium chloride, magnesium acetate, magnesium phosphate, magnesium iodide, magnesium fluoride, magnesium bromide, or a combination thereof.

[0178] Disclosed herein are methods of assaying for a target nucleic acid as described herein wherein a signal is detected. For example, a method of assaying for a target nucleic acid in a sample comprises 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 protein-nucleic acids of a population of 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. As another example, a method of assaying for a target nucleic acid in a sample, for example, comprises: a) 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 (e.g., a nucleic acid from a coronavirus such as SARS-CoV-2) 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; b) contacting the complex to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, 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. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.

[0179] A programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid (e.g., a nucleic acid from a coronavirus such as SARS-CoV-2). 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 nucleic acids by the activated programmable nuclease, such as trans cleavage of reporters with a detection moiety. Once the reporter is cleaved by the activated programmable nuclease, the detection moiety can be released from the reporter and can generate a signal. The detection moiety capable of generating the detectable signal can be immobilized on a support medium for detection. The signal can be visualized to assess whether a target nucleic acid comprises a modification.

[0180] Often, the signal is a colorimetric signal or a signal visible by eye. In some instances, the signal is fluorescent, electrical, chemical, electrochemical, or magnetic. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, the detectable signal is a colorimetric signal or a signal visible by eye. In some instances, the detectable signal is fluorescent, electrical, chemical, electrochemical, or magnetic. In some cases, the first detection signal is generated by binding of the detection moiety to the capture molecule in the detection region, where the first detection signal indicates that the sample contained the target nucleic acid. Sometimes the system is capable of detecting more than one type of target nucleic acid, wherein the system comprises more than one type of guide nucleic acid and more than one type of reporter. In some cases, the detectable signal is generated directly by the cleavage event. Alternatively, or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal is a colorimetric or color-based signal. In some cases, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium. In some cases, the second detectable signal is generated in a spatially distinct location than the first generated signal.

[0181] In some cases, the threshold of detection, for a subject method of detecting a single-stranded target nucleic acid in a sample, is less than or equal to 10 nM. The term "threshold of detection" is used herein to describe the minimal amount of target nucleic acid that must be present in a sample in order for detection to occur. For example, when a threshold of detection is 10 nM, then a signal can be detected when a target nucleic acid is present in the sample at a concentration of 10 nM or more. In some cases, the threshold of detection is less than or equal to 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM, 0.0005 nM, 0.0001 nM, 0.00005 nM, 0.00001 nM, 10 pM, 1 pM, 500 fM, 250 fM, 100 fM, 50 fM, 10 fM, 5 fM, 1 fM, 500 attomole (aM), 100 aM, 50 aM, 10 aM, or 1 aM. In some cases, the threshold of detection is in a range of from 1 aM to 1 nM, 1 aM to 500 pM, 1 aM to 200 pM, 1 aM to 100 pM, 1 aM to 10 pM, 1 aM to 1 pM, 1 aM to 500 fM, 1 aM to 100 fM, 1 aM to 1 fM, 1 aM to 500 aM, 1 aM to 100 aM, 1 aM to 50 aM, 1 aM to 10 aM, 10 aM to 1 nM, 10 aM to 500 pM, 10 aM to 200 pM, 10 aM to 100 pM, 10 aM to 10 pM, 10 aM to 1 pM, 10 aM to 500 fM, 10 aM to 100 fM, 10 aM to 1 fM, 10 aM to 500 aM, 10 aM to 100 aM, 10 aM to 50 aM, 100 aM to 1 nM, 100 aM to 500 pM, 100 aM to 200 pM, 100 aM to 100 pM, 100 aM to 10 pM, 100 aM to 1 pM, 100 aM to 500 fM, 100 aM to 100 fM, 100 aM to 1 fM, 100 aM to 500 aM, 500 aM to 1 nM, 500 aM to 500 pM, 500 aM to 200 pM, 500 aM to 100 pM, 500 aM to 10 pM, 500 aM to 1 pM, 500 aM to 500 fM, 500 aM to 100 fM, 500 aM to 1 fM, 1 fM to 1 nM, 1 fM to 500 pM, 1 fM to 200 pM, 1 fM to 100 pM, 1 fM to 10 pM, 1 fM to 1 pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fM to 200 pM, 10 fM to 100 pM, 10 fM to 10 pM, 10 fM to 1 pM, 500 fM to 1 nM, 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, from 1 pM to 1 nM, 1 pM to 500 pM, 1 pM to 200 pM, 1 pM to 100 pM, or 1 pM to 10 pM. In some cases, the threshold of detection in a range of from 800 fM to 100 pM, 1 pM to 10 pM, 10 fM to 500 fM, 10 fM to 50 fM, 50 fM to 100 fM, 100 fM to 250 fM, or 250 fM to 500 fM. In some cases, the minimum concentration at which a single-stranded target nucleic acid is detected in a sample is in a range of from 1 aM to 1 nM, 10 aM to 1 nM, 100 aM to 1 nM, 500 aM to 1 nM, 1 fM to 1 nM, 1 fM to 500 pM, 1 fM to 200 pM, 1 fM to 100 pM, 1 fM to 10 pM, 1 fM to 1 pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fM to 200 pM, 10 fM to 100 pM, 10 fM to 10 pM, 10 fM to 1 pM, 500 fM to 1 nM, 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, 1 pM to 1 nM, 1 pM to 500 pM, from 1 pM to 200 pM, 1 pM to 100 pM, or 1 pM to 10 pM. In some cases, the minimum concentration at which a single-stranded target nucleic acid can be detected in a sample is in a range of from 1 aM to 100 pM. In some cases, the minimum concentration at which a single-stranded target nucleic acid can be detected in a sample is in a range of from 1 fM to 100 pM. In some cases, the minimum concentration at which a single-stranded target nucleic acid can be detected in a sample is in a range of from 10 fM to 100 pM. In some cases, the minimum concentration at which a single-stranded target nucleic acid can be detected in a sample is in a range of from 800 fM to 100 pM. In some cases, the minimum concentration at which a single-stranded target nucleic acid can be detected in a sample is in a range of from 1 pM to 10 pM. In some cases, the devices, systems, fluidic devices, kits, and methods described herein detect a target single-stranded nucleic acid in a sample comprising a plurality of nucleic acids such as a plurality of non-target nucleic acids, where the target single-stranded nucleic acid is present at a concentration as low as 1 aM, 10 aM, 100 aM, 500 aM, 1 fM, 10 fM, 500 fM, 800 fM, 1 pM, 10 pM, 100 pM, or 1 pM.

[0182] In some cases, the devices, systems, fluidic devices, kits, and methods described herein detect a target single-stranded nucleic acid (e.g., a nucleic acid from a coronavirus such as SARS-CoV-2) in a sample where the sample is contacted with the reagents for a predetermined length of time sufficient for the trans cleavage to occur or cleavage reaction to reach completion. In some cases, the devices, systems, fluidic devices, kits, and methods described herein detect a target single-stranded nucleic acid in a sample where the sample is contacted with the reagents for no greater than 60 minutes. Sometimes the sample is contacted with the reagents for no greater than 120 minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute. Sometimes the sample is contacted with the reagents for at least 120 minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes. In some cases, the devices, systems, fluidic devices, kits, and methods described herein can detect a target nucleic acid in a sample in less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 35 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, or less than 5 minutes.

[0183] When a guide nucleic acid binds to a target nucleic acid (e.g., a nucleic acid from a coronavirus such as SARS-CoV-2), the programmable nuclease's trans cleavage activity can be initiated, and reporters can be cleaved, resulting in the detection of fluorescence. Some methods as described herein can a method of assaying for a target nucleic acid in a sample comprises 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 protein-nucleic acids of a population of 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. The cleaving of the reporter using the programmable nuclease may cleave with an efficiency of 50% as measured by a change in a signal that is calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric, as non-limiting examples. Some methods as described herein can be a method of detecting a target nucleic acid in a sample comprising contacting the sample comprising the target nucleic acid with a guide nucleic acid targeting a target nucleic acid segment, a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment, a single-stranded reporter comprising a detection moiety, wherein the reporter is capable of being cleaved by the activated programmable nuclease, thereby generating a first detectable signal, cleaving the single-stranded reporter using the programmable nuclease that cleaves as measured by a change in color, and measuring the first detectable signal on the support medium. The cleaving of the single-stranded reporter using the programmable nuclease may cleave with an efficiency of 50% as measured by a change in color. In some cases, the cleavage efficiency is at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% as measured by a change in color. The change in color may be a detectable colorimetric signal or a signal visible by eye. The change in color may be measured as a first detectable signal. The first detectable signal can be detectable within 5 minutes of contacting the sample comprising the target nucleic acid with a guide nucleic acid targeting a target nucleic acid segment, a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment, and a single-stranded reporter comprising a detection moiety, wherein the reporter is capable of being cleaved by the activated nuclease. The first detectable signal can be detectable within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, or 120 minutes of contacting the sample.

[0184] In some cases, the methods, reagents, and devices described herein detect a target nucleic acid with a programmable nuclease and a single-stranded reporter in a sample where the sample is contacted with the reagents for a predetermined length of time sufficient for trans cleavage of the single-stranded reporter. For example, a programmable nuclease is LbuCas13a that detects a target nucleic acid and a single-stranded reporter comprises two adjacent uracil nucleotides with a green detectable moiety that is detected upon cleavage. As another example, a programmable nuclease is LbaCas13a that detects a target nucleic acid and a single-stranded reporter comprises two adjacent adenine nucleotides with a red detectable moiety that is detected upon cleavage. The target nucleic acid may be a single-stranded nucleic acid (e.g., a single-stranded DNA (ssDNA) or a single-stranded RNA), or the target nucleic acid may be a double-stranded nucleic acid (e.g., a double-stranded DNA (dsDNA) or a double-stranded RNA).Buffers for Detection of Nucleic Acids

[0185] The reagents described herein can also include buffers, which are compatible with the devices, systems, fluidic devices, kits, and methods disclosed herein. These buffers are compatible with the other reagents, samples, and support mediums as described herein for detection of an ailment, such as a disease, including those caused by viruses such as influenza. The methods described herein can also include the use of buffers, which are compatible with the methods disclosed herein.

[0186] A buffer may be configured to support multiple reactions or processes. A buffer may be configured to enable fast reaction rates for multiple types of reactions. For example, a buffer may enable a reaction to achieve a rate within 25%, 50%, 75%, or 90% of the fastest reported rate for that reaction at a specified temperature. The specified temperature may be between 0 and 10 °C, 10 and 20 °C, 20 and 30 °C, 30 and 40 °C, 40 and 50 °C, 50 and 60 °C, 60 and 70 °C, 70 and 80 °C, 80 and 90 °C, or higher than 90 °C. A buffer may enable amplification and DETECTR reactions to each reach completion (e.g., consume at least 80% of a limiting reagent) in less than two hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 20 minutes, or less than 15 minutes. A buffer may enable fast reaction rates for 2, 3, 4, or 5 or more types of reactions, wherein each reaction comprises one or more targets. For example, a buffer may enable fast reaction rates for amplification and DETECTR reactions, and may comprise amplification and DETECTR reagents each targeting 100 nucleic acid sequences.

[0187] Time to completion may be measured by the consumption of a reagent. For example, a single-buffer comprising amplification and DETECTR reagents may consume (e.g., subject to transcollateral cleavage) at least 50 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising amplification and DETECTR reagents may consume at least 20 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising amplification and DETECTR reagents may consume at least 10 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising amplification and DETECTR reagents may consume at least 5 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising amplification and DETECTR reagents may consume at least 1 nM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid. A single-buffer comprising amplification and DETECTR reagents may consume at least 500 pM of reporters within 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, or 15 minutes of addition of 10000 copies, 5000 copies, 2000 copies, 1000 copies, 500 copies, 300 copies, 200 copies, 100 copies, 50 copies, or 10 copies of a target nucleic acid.

[0188] A buffer may also be configured to maximize the stability of a reagent (e.g., a programmable nuclease or a target nucleic acid) or reaction species (e.g., a DNA molecule produced by reverse transcription of a target RNA molecule). For example, a buffer may stabilize RNA so that its average half-life is 1.5 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes, 5 minutes, 3 minutes, 2 minutes, or 1 minute at room temperature. A buffer may stabilize a reagent or enzyme for long-term storage. A buffer of the present disclosure may enable stable, long-term storage for a set of reagents, such as a composition comprising amplification and DETECTR reagents. A central challenge to the design of such a buffer stems from different proteins having different optimal conditions for stability. Optimal conditions for two proteins may comprise different osmolarities, viscosities, dielectric constants, pH, lipid content, detergent content, proportions of non-aqueous solvents (e.g., methanol, ethanol, acetone, acetonitrile, or a halomethane such as trichloromethane), or densities. The present disclosure provides buffers that are optimized to simultaneously stabilize a plurality of different proteins, such as a polymerase and a programmable nuclease (e.g., CRISPR-Cas enzyme). Enzymes for amplification and CRISPR-Cas reactions may have half-lives of at least three months, two months, one month, two weeks, one week, five days, 3 days, 2 days, 1 day, 12 hours, 8 hours, 6 hours, 4 hours, or 3 hours, 2 hours, or 1 hours at room temperature...

Claims

1. A system for detecting a target nucleic acid, comprising a buffer comprising: i. amplification reagents for an amplification reaction targeting the target nucleic acid; and ii. detection reagents for a detection reaction targeting the target nucleic acid; wherein the amplification reagents comprise one or more oligonucleotide primers, and a DNA polymerase; wherein the detection reagents comprise a programmable nuclease, a non-naturally occurring guide nucleic acid, and reporters; wherein the non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid or DNA amplicons thereof; wherein the amplification reagents are present in amounts effective to amplify the target nucleic acid in a test sample to produce DNA amplicons of the target nucleic acid; wherein the programmable nuclease and non-naturally occurring guide nucleic acid form a complex in the buffer that is activated upon binding one of the DNA amplicons to induce detectable transcollateral cleavage of the reporters; and wherein the buffer is a lysis buffer.

2. The system of claim 1, wherein (a) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (b) at least 5 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (c) at least 10 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 5000 copies of the target nucleic acid to the system; (d) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system; or (e) at least 1 nM of the reporters undergo transcollateral cleavage within one hour of addition of at least 1000 copies of the target nucleic acid to the system.

3. The system of claim 1 or claim 2, wherein the amplification reagents comprise reagents for isothermal amplification; optionally wherein the amplification reagents comprise reagents for transcription mediated amplification (TMA), helicase dependent amplification (HDA), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), 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).

4. The system of claim 1, further comprising an activator for the amplification reaction; optionally wherein the activator for the amplification reaction comprises a magnesium or calcium salt.

5. The system of any one of claims 1-4, wherein the programmable nuclease comprises at least 60% sequence identity to SEQ ID NO: 18-170, 221-268 or 397-423.

6. The system of any one of claims 1-5, wherein the programmable nuclease is a CRISPR-Cas enzyme, optionally a Type V CRISPR / Cas effector protein.

7. The system of any one of claims 1-6, further comprising a reverse transcriptase, an oligonucleotide primer, and dNTPs for reverse transcribing the target nucleic acid.

8. The system of any one of claims 1-7, wherein the buffer comprises a pH of 7.5 to 8.5, at least 10 mM of a buffering agent, at least 1 mM ammonium acetate, at least 10 mM potassium acetate, at least 2.5 mM magnesium acetate, and at least 0.5% glycerol; optionally wherein: (a) the buffer comprises a pH of 7.7 to 8.3, or a pH of 7.85 to 8.15; (b) the buffering agent comprises HEPES, imidazole, TRIS-HCl, or phosphate; and / or (c) the buffer further comprises at least 0.05% by volume of a detergent, optionally wherein the detergent comprises Tween 20.

9. The system of any one of claims 1-7, wherein the buffer comprises a pH of 7.5 to 8.5, at least 5 mM of a buffering agent, at least 20 mM potassium acetate, at least 2.5 mM magnesium acetate, and at least 0.5% glycerol; optionally wherein: (a) the buffer comprises a pH of 7.7 to 8.3, or a pH of 7.85 to 8.15; (b) the buffering agent comprises phosphate or TRIS-HCl; (c) the buffer comprises at least 1 mM ammonium sulfate; and / or (d) the buffer comprises at least 0.05% by volume of a detergent, optionally wherein the detergent comprises Tween 20.

10. The system of any one of claims 1-7, wherein the buffer comprises a pH of 7.25 to 8.75, at least 5 mM of a buffering agent, at least 7.5 mM potassium acetate, at least 1 mM magnesium acetate, and at least 0.5% glycerol; optionally wherein: (a) the buffering agent comprises phosphate; (b) the buffer comprises a pH of 7.5 to 8.5, or a pH of 7.75 to 8.25; (c) the buffer further comprises at least 1 mM ammonium sulfate; and / or (d) the buffer further comprises at least 0.05% by volume of a detergent, optionally wherein the detergent comprises Tween 20.

11. The system of claim 1 or 2, further comprising a circular template with internal complementarity formed from a single polynucleotide strand, wherein: (a) the circular template comprises a first portion with complementarity to one of the one or more oligonucleotide primers and a second portion with complementarity to a portion of the target nucleic acid; (b) the internal complementarity comprises part of the first portion and part of the second portion; (c) the second portion has a total length that is longer than a combined length of the first portion and second portion that are within the internal complementarity; and (d) the circular template undergoes a conformational change upon hybridization to the target nucleic acid to expose the first portion to hybridization to the oligonucleotide primer.

12. The system of claim 1 or 2, further comprising a circular template, wherein: (a) the circular template comprises a first portion with complementarity to one of the one or more oligonucleotide primers and a second portion with complementarity to the target nucleic acid; (b) the oligonucleotide primer complementary to the first portion comprises a blocking motif at its 3' end; and (c) the oligonucleotide primer complementary to the first portion undergoes cleavage to remove the blocking motif by the programmable nuclease in the presence of the target nucleic acid.

13. The system of any one of claims 1-12, further comprising a polymer matrix, wherein the polymer matrix is complexed with the reporters; optionally wherein: (a) the polymer matrix is formed from copolymerization of at least a first plurality of monomers with the reporters, and / or (b) the polymer matrix comprises a hydrogel.

14. A method of assaying for a target nucleic acid in a sample, the method comprising: a. amplifying a portion of the target nucleic acid with a DNA polymerase to produce DNA amplicons of the target nucleic acid; b. forming a complex comprising one of the DNA amplicons, a programmable nuclease, and a non-naturally occurring guide nucleic acid that hybridizes to a segment of the DNA amplicon, thereby activating the programmable nuclease; c. cleaving reporters with the activated programmable nuclease; and d. detecting a change in a signal, wherein the change in the signal is produced by cleavage of the reporters; wherein the target nucleic acid and reagents for the amplifying and cleaving are present in the same reaction volume; wherein the method further comprises lysing a cell or virus comprising the target nucleic acid and wherein the lysing is performed in the same reaction volume as the amplifying and the cleaving.

15. A system for detecting a target nucleic acid, comprising reagents in a buffer, wherein: (a) the reagents comprise hairpin polynucleotides, programmable nucleases, non-naturally occurring guide nucleic acids, and reporters; (b) each hairpin polynucleotide comprises one or more RNA loops, a first portion comprising DNA, and a second portion joined to the first portion by one of the one or more RNA loops; (c) each non-naturally occurring guide nucleic acid comprises a sequence that hybridizes to a segment of the target nucleic acid; (d) in each hairpin polynucleotide, the second portion of the hairpin polynucleotide hybridizes to a segment of the first portion; (e) the programmable nucleases and non-naturally occurring guide nucleic acids form complexes in the buffer that are activated upon binding the target nucleic acid; (f) an activated programmable nuclease is effective to induce (i) transcollateral cleavage of the one or more RNA loops, and (ii) detectable transcollateral cleavage of the reporters; and (g) cleavage of the one or more RNA loops of one of the hairpin polynucleotides is effective to release the first portion of the hairpin polynucleotide to hybridize with one of the non-naturally occurring guide nucleic acids and form a further activated programmable nuclease.

16. A method of assaying for a target nucleic acid in a sample, the method comprising the following steps in a single reaction volume: (a) forming a complex comprising the target nucleic acid, a first programmable nuclease, and a first non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, thereby activating the programmable nuclease; (b) cleaving a hairpin polynucleotide of a plurality of hairpin polynucleotides with the activated programmable nuclease, wherein each hairpin polynucleotide comprises (i) one or more RNA loops that are cleaved, (i) a first portion comprising DNA, and (iii) a second portion joined to the first portion by one of the one or more RNA loops, wherein the second portion is hybridized to a segment of the first portion; (c) forming a second complex comprising the first portion of the cleaved hairpin polynucleotide, a second programmable nuclease, and a second non-naturally occurring guide nucleic acid that hybridizes to the first portion of the cleaved hairpin, thereby activating the second programmable nuclease; (d) cleaving reporters with the activated first or second programmable nuclease; and (e) detecting a change in a signal, wherein the change in the signal is produced by cleavage of the reporters.