Patent Application
20240384332
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Mar. 29, 2024, is named 203477-747301_US_SL.xml and is 2,182,193 bytes in size.
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. Challenges in deploying such devices include developing methods to immobilize diagnostic assay components on surfaces without compromising the performance of the assay, as well as performing amplification of samples without significant additional instrumentation.
In an aspect, the present disclosure provides a programmable nuclease diagnostic device that may comprise a sample interface configured to receive a sample that may comprise at least one sequence of interest; a channel in fluid communication with the sample interface and a detection chamber, said channel comprising one or more movable mechanisms to separate the sample into a plurality of droplets, wherein said detection chamber is configured to receive and contact the plurality of droplets with at least one programmable nuclease probe disposed on a surface of said detection chamber, wherein said at least one programmable nuclease probe may comprise a guide nucleic acid complexed with a programmable nuclease; and a plurality of sensors that determine a presence of said at least one sequence of interest by detecting a signal produced upon cleavage of a target nucleic acid region of said at least one sequence of interest by said at least one programmable nuclease probe. In some embodiments, the cleavage of said target nucleic acid region occurs after a complementary binding of said target nucleic acid region to said guide nucleic acid of said at least one programmable nuclease probe. In some embodiments, the programmable nuclease probe may comprise a CRISPR/Cas enzyme. In some embodiments, the guide nucleic acid may comprise a guide RNA. In some embodiments, the one or more movable mechanisms comprise one or more valves configured to restrict flow through one or more sections of the channel. In some embodiments, the one or more movable mechanisms comprise a plunger or a bristle that is configured to restrict flow through one or more sections of the channel. In some embodiments, the one or more movable mechanisms are operatively coupled to a power source that is integrated with or insertable into the device. In some embodiments, the power source may comprise a battery. In some embodiments, the device may comprise a physical filter to filter one or more particles from the sample that do not comprise the sequence of interest. In some embodiments, the device may comprise a sample preparation chamber. In some embodiments, the sample preparation chamber may comprise a lysing agent. In some embodiments, the sample preparation chamber may comprise a heating unit configured for heat inactivation. In some embodiments, the sample preparation chamber may comprise one or more reagents for nucleic acid purification. In some embodiments, the channel may comprise a plurality of heating elements and a plurality of heat sinks for amplifying the at least one sequence of interest or a portion thereof. In some embodiments, the plurality of heating elements and the plurality of heat sinks are configured to perform one or more thermocycling operations on the plurality of droplets. In some embodiments, the device may comprise a plurality of programmable nuclease probes comprising different guide RNAs. In some embodiments, the signal is associated with a physical, chemical, or electrochemical change or reaction. In some embodiments, the signal may comprise an optical signal. In some embodiments, the signal may comprise a fluorescent or colorimetric signal. In some embodiments, the signal may comprise a potentiometric or amperometric signal. In some embodiments, the signal may comprise a piezo-electric signal. In some embodiments, the signal is associated with a change in an index of refraction of a solid or gel volume in which said at least one programmable nuclease probe is disposed.
In another aspect, the present disclosure provides a device that may comprise a sample interface configured to receive a sample that may comprise one or more target sequences of interest; one or more channels comprising one or more movable mechanisms to separate the sample into partitioned samples, wherein the one or more channels are in fluid communication with the sample interface and a reaction chamber that is configured to receive and contact the partitioned samples with an enzyme, reagent, or programmable detection agent that is configured to cleave a nucleic acid of said one or more target sequences of interest; and a plurality of sensors for determining a presence of the one or more target sequences of interest by detecting one or more reporters released upon said cleavage of said nucleic acid. In some embodiments, the programmable detection agent may comprise a CRISPR/Cas enzyme. In some embodiments, the one or more target sequences of interest comprise a sequence of nucleic acids comprising said nucleic acid. In some embodiments, the one or more movable mechanisms comprise a plurality of valves configured to restrict flow in a first direction through the one or more channels towards the sample interface. In some embodiments, the plurality of valves are configured to selectively permit flow in a second direction through the one or more channels towards the reaction chamber. In some embodiments, a first valve and a second valve of the plurality of valves are configured to physically, fluidically, or thermally isolate a first portion of the sample from a second portion of the sample when the first valve and the second valve are in a closed state. In some embodiments, the one or more channels comprise a plurality of heating elements and a plurality of heat sinks to perform thermocycling on the partitioned samples. In some embodiments, a first heating element of the plurality of heating elements and a first heat sink of the plurality of heat sinks are positioned between a first movable mechanism and a second movable mechanism of the one or more movable mechanisms. In some embodiments, the reporter may comprise a nucleic acid and a detection moiety. In some embodiments, the reporter may comprise at least one ribonucleotide or at least one deoxyribonucleotide. In some embodiments, the reporter may comprise a DNA nucleic acid or an RNA nucleic acid. In some embodiments, the device may comprise a telemedicine unit configured to provide one or more detection results to a computing unit that is remote from the device, wherein the one or more detection results indicate a presence or an absence of a target nucleic acid of interest in the sample.
In another aspect, the present disclosure provides a method for target detection, comprising: contacting a sample with any of the devices described herein; and detecting a presence or an absence of one or more genes of interest in said sample. In some embodiments, the method may comprise generating one or more detection results indicating the presence or the absence of the one or more genes of interest in the sample. In some embodiments, the method may comprise transmitting the one or more detection results to a remote computing unit. In some embodiments, the remote computing unit may comprise a mobile device. In another aspect, the present disclosure provides a method for target detection, comprising: providing a sample comprising at least one gene of interest; separating the sample into a plurality of sub-samples using one or more movable mechanisms; receiving the plurality of sub-samples in a detection chamber and contacting the plurality of sub-samples with at least one programmable nuclease probe disposed on a surface of said detection chamber, wherein said at least one programmable nuclease probe may comprise a guide nucleic acid complexed with a programmable nuclease; and using a plurality of sensors to determine a presence or an absence of said at least one gene of interest by detecting a signal produced upon cleavage of a target nucleic acid region in said at least one gene of interest by said at least one programmable nuclease probe. In some embodiments, the method may comprise amplifying the at least one gene of interest after separating the sample into a plurality of sub-samples. In some embodiments, the method may comprise amplifying the at least one gene of interest before the plurality of sub-samples are received in the detection chamber. In some embodiments, amplifying the at least one gene of interest may comprise using a plurality of heating elements and a plurality of heat sinks to perform thermocycling on the plurality of sub-samples. In some embodiments, the programmable nuclease probe may comprise a CRISPR/Cas enzyme. In some embodiments, the guide nucleic acid may comprise a guide RNA. In some embodiments, the one or more movable mechanisms comprise one or more valves configured to restrict flow through one or more sections of the channel. In some embodiments, the one or more movable mechanisms comprise a plunger or a bristle that is configured to restrict flow through one or more sections of the channel. In some embodiments, the method may comprise using a physical filter to filter one or more particles from the sample that do not comprise the at least one gene of interest. In some embodiments, the method may comprise lysing the sample before detecting the at least one gene of interest. In some embodiments, the method may comprise performing enzyme (e.g., PK) inactivation on the sample. In some embodiments, the method may comprise performing heat inactivation on the sample. In some embodiments, the method may comprise performing nucleic acid purification on the sample. In some embodiments, the method may comprise contacting the plurality of sub-samples with a plurality of programmable nuclease probes comprising different guide RNAs. In some embodiments, the signal is associated with a physical, chemical, or electrochemical change or reaction. In some embodiments, the signal is selected from the group consisting of an optical signal, a fluorescent signal, a colorimetric signal, a potentiometric signal, an amperometric signal, and a piezo-electric signal. In some embodiments, the signal is associated with a change in an index of refraction of a solid or gel volume in which said at least one programmable nuclease probe is disposed. In some embodiments, the method may comprise using the signal to detect pathogenic viruses, pathogenic bacteria, pathogenic worms, pathogenic fungi, or cancer cells. In some embodiments, the pathogenic viruses are selected from the group consisting of respiratory viruses, adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, SARS-CoV, SARS-CoV-2, MERS, gastrointestinal viruses, noroviruses, rotaviruses, astroviruses, exanthematous viruses, hepatic viral diseases, cutaneous viral diseases, herpes, hemorrhagic viral diseases, Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, neurologic viruses, polio, viral meningitis, viral encephalitis, rabies, sexually transmitted viruses, HIV, HPV, immunodeficiency viruses, influenza virus, dengue virus, West Nile virus, herpes virus, yellow fever virus, Hepatitis Virus C, Hepatitis Virus A, Hepatitis Virus B, and papillomavirus. In some embodiments, the method may comprise amplifying or modifying the signal using a physical or chemical interaction between a reporter that is released upon cleavage and another material, entity, or molecular species in the detection chamber. In some embodiments, the devices of the present disclosure are configured to detect pathogenic viruses, pathogenic bacteria, pathogenic worms, pathogenic fungi, or cancer cells based on the signal. In some embodiments, the pathogenic viruses are selected from the group consisting of respiratory viruses, adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, SARS-CoV, SARS-CoV-2, MERS, gastrointestinal viruses, noroviruses, rotaviruses, astroviruses, exanthematous viruses, hepatic viral diseases, cutaneous viral diseases, herpes, hemorrhagic viral diseases, Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, neurologic viruses, polio, viral meningitis, viral encephalitis, rabies, sexually transmitted viruses, HIV, HPV, immunodeficiency viruses, influenza virus, dengue virus, West Nile virus, herpes virus, yellow fever virus, Hepatitis Virus C, Hepatitis Virus A, Hepatitis Virus B, and papillomavirus. In some embodiments, the detection chamber or the reaction chamber of the device may comprise another material, entity, or molecular species that is configured to physically or chemically interact or react with a reporter that is released upon cleavage to amplify or modify the signal. In some embodiments, the sequence of interest may comprise a biological sequence. The biological sequence can comprise a nucleic acid sequence or an amino acid sequence. In some embodiments, the sequence of interest is associated with an organism of interest, a disease of interest, a disease state of interest, a phenotype of interest, a genotype of interest, or a gene of interest.
In another aspect, a reaction chamber may comprise a surface wherein a probe comprising at least one of said enzyme, said reagent, said programmable detection reagent, a programmable nuclease, a guide nucleic acid, a reporter or a combination thereof, and wherein said probe is immobilized to said surface by a linkage. In some embodiments, a linkage may comprise a surface functionality and a probe functionality. In some embodiments, a surface functionality is disposed on said surface. In some embodiments, a surface functionality is streptavidin. In some embodiments, an amino acid residue of said programmable nuclease is connected to said surface by said linkage. In some embodiments, an amino acid residue is modified with said probe functionality. In some embodiments, a probe functionality is biotin. In some embodiments, a guide nucleic acid is connected to said surface by said linkage. In some embodiments, a guide nucleic acid is modified at the 3′ end or 5′ end with said probe functionality. In some embodiments, a reporter is connected to said surface by said linkage. In some embodiments, a reporter may comprise at least one of said nucleic acid, said probe functionality, a detection moiety, a quencher or a combination thereof. In some embodiments, a reporter is configured for said detection moiety to remain immobilized to said surface and said quencher to be released into solution upon cleavage of said reporter. In some embodiments, a reporter is configured for said quencher to remain immobilized to said surface and for said detection moiety to be released into solution, upon cleavage of said reporter.
In one aspect, the present disclosure provides a composition comprising a plurality of reporters. In some embodiments, (a) each reporter of the plurality of reporters comprises one or more enzymes conjugated to a linker; (b) the linker comprises one or more nucleic acid sections; and (c) the linker (i) comprises a functionality for immobilization to a support, or (ii) is conjugated to the support. Conjugations can be covalent or non-covalent. In some embodiments, the one or more enzymes is a single enzyme, such as a single horseradish peroxidase (HRP). In some embodiments, the one or more enzymes comprises a poly-HRP comprising a plurality of HRP enzymes. In some embodiments, the plurality of HRP enzymes of the poly-HRP are complexed with streptavidin, and the linker comprises a biotin for conjugation with the poly-HRP. In some embodiments, the one or more nucleic acid sections comprise one or more sections of DNA, one or more sections of RNA, or a combination thereof. In some embodiments, the one or more nucleic acid sections consist of DNA. In some embodiments, the one or more nucleic acid sections consist of RNA. In some embodiments, the one or more nucleic acid sections comprise one or more sections of DNA and one or more sections of RNA. In some embodiments, the one or more nucleic acid sections are single-stranded. In some embodiments, the one or more nucleic acid sections have a combined length of at least 20, 30, 40, or 50 nucleotides. In some embodiments, at least one of the one or more nucleic acid sections is a substrate for cleavage by a programmable nuclease (e.g., any of the programmable nucleases described herein). In some embodiments, the linker comprises (a) a first portion comprising a non-naturally occurring guide nucleic acid, and (b) a second portion comprising a nucleic acid section that is a substrate for cleavage by a programmable nuclease, and the one or more enzymes (e.g., HRP) are conjugated to the linker via the second portion. In some embodiments, the linker further comprises one or more hydrocarbon chains. In some embodiments, the one or more hydrocarbon chains comprises a hydrocarbon chain between the one or more nucleic acid sections and the support. In some embodiments, the one or more hydrocarbon chains comprises a hydrocarbon chain between the one or more nucleic acid sections and the one or more enzymes. In some embodiments, the support is a bead, a hydrogel, or the surface of a container (e.g., a well or a tube). In some embodiments, the composition further comprises a programmable nuclease (e.g., any of the programmable nucleases described herein).
In one aspect, the present disclosure provides methods for detecting a target nucleic acid in a sample using a composition as described herein. In some embodiments, the method comprises (a) contacting the sample with a composition described herein to produce a reaction fluid, wherein (i) the linker is conjugated to the support, (ii) the composition further comprises a guide nucleic acid configured to bind to the target nucleic acid, and (iii) the programmable nuclease cleaves the linker in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (b) separating the reaction fluid from the support; (c) contacting the separated reaction fluid with a substrate of the one or more enzymes, wherein presence of the one or more enzymes in the fluid produces a detectable signal from the enzyme substrate; and (d) detecting the detectable signal. In some embodiments, the method comprises (a) contacting the sample with a composition described herein to produce a reaction fluid, wherein (i) the composition further comprises a programmable nuclease and a guide nucleic acid, (ii) the guide nucleic acid is configured to bind to the target nucleic acid, and (iii) the programmable nuclease cleaves the linker in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (b) contacting the reaction fluid with a plurality of beads, wherein each bead reacts with a plurality of the functionalities, thereby immobilizing the functionalities to the beads; (c) separating the reaction fluid from the beads; (d) contacting the separated reaction fluid with a substrate of the one or more enzymes, wherein presence of the one or more enzymes in the fluid produces a detectable signal from the enzyme substrate; and (e) detecting the detectable signal. In some embodiments, the method further comprises concentrating the one or more enzymes prior to contacting the one or more enzymes with the substrate, wherein the concentrating comprises separating the one or more enzymes from at least a portion of the separated reaction fluid. In some embodiments, concentrating comprises capture of the enzyme on a support. In some embodiments, contacting the separated reaction fluid with the substrate of the one or more enzymes comprises contacting the enzyme on the support with the substrate 2, 3, 4, 5, or more times. In some embodiments, the detectable signal comprises a change in an absorbance, and detection comprises detecting or measuring the change in absorbance.
In various aspects described herein, a reporter is connected to said surface by a linkage. In some embodiments, a linkage may comprise a surface functionality and a probe functionality. In some embodiments, a surface functionality is disposed on said surface and said reporter may comprise said probe functionality.
In certain aspects, described herein are embodiments of a method for target detection, comprising: providing a sample comprising at least one sequence of interest; separating the sample into a plurality of sub-samples using one or more movable mechanisms; receiving the plurality of sub-samples in a detection chamber and contacting the plurality of sub-samples with at least one probe, wherein said at least one probe is connected to a surface of said detection chamber by a linkage, wherein said at least one probe may comprise a programmable nuclease, a guide nucleic acid, a reporter or a combination thereof; and using a plurality of sensors to determine a presence of said at least one sequence of interest by detecting a signal produced upon cleavage of said reporter by said programmable nuclease.
Described herein are various embodiments of a device comprising: a sample interface configured to receive a sample that may comprise one or more target sequences of interest; one or more channels comprising one or more movable mechanisms to separate said sample into partitioned samples, wherein said one or more channels are in fluid communication with said sample interface and a reaction chamber comprising a surface, wherein at least one probe may comprise a programmable nuclease, a guide nucleic acid, a reporter or a combination thereof, wherein said at least one probe is connected to said surface by a linkage; and a plurality of sensors for determining a presence of said one or more target sequences of interest by detecting a signal emitted upon cleavage of said reporter by said programmable nuclease.
Described herein are various devices for detecting a target nucleic acid, comprising: a sample interface configured to receive a sample comprising a target nucleic acid; a reaction chamber (e.g., a heating region) in fluid communication with the sample interface and configured to amplify the sample received via the sample interface; a detection region in fluid communication with the heating region; and a programmable nuclease probe disposed within the sample interface, the heating region, and/or the detection region, wherein a signal is produced via selective binding between the programmable nuclease probe and the target nucleic acid within the heating region, the sample interface, and/or the detection region, wherein the detection region is configured to detect the signal corresponding to a presence of the target nucleic acid, and wherein the presence or absence of the target nucleic acid is determined within a time of less than 30 minutes after the sample is received at the sample interface. In some embodiments, a reagent mix comprising amplification reagents. In some embodiments, the reagent mix is lyophilized. In some embodiments, the reagent mix is located in a region of the device that is in fluid communication with both the sample interface and the heating region. In some embodiments, the reagent mix is located within the sample interface, the heating region, the detection region, and/or a region between the sample interface and the heating region. In some embodiments, the heating region comprises amplification reagents. In some embodiments, the sample is amplified via Loop-Mediated Isothermal Amplification (LAMP). In some embodiments, the heating region is configured to maintain an isothermal, or non-cycled temperature profile. In some embodiments, the isothermal, or non-cycled temperature profile is between about 30° C. to about 60° C. In some embodiments, the isothermal, or non-cycled temperature profile is about 55° C. to about 60° C. In some embodiments, the sample interface comprises a compartment configured to receive a swab containing the sample. In some embodiments, the compartment comprises a scraper configured to transfer the sample from the swab to the device. In some embodiments, the compartment contains a interface solution configured to extract the sample from the swab. In some embodiments, the interface solution comprises a buffer solution or a lysis buffer solution. In some embodiments, the sample interface is configured to receive the sample from a swab via pipetting. In some embodiments, the sample interface comprises a compartment configured to receive the sample from a container containing the sample. In some embodiments, the container comprises a syringe. In some embodiments, the syringe interface comprises an opening for receiving the sample therethrough. In some embodiments, the syringe interface opening is in fluid communication with i) the heating region, and/or ii) another compartment that is in fluid communication with the heating region. In some embodiments, the sample interface is configured to receive the sample as a fluid. In some embodiments, the heating region and detection region are disposed on the same location on the device. In some embodiments, the heating region and the detection region are disposed within a same compartment of the device. In some embodiments, the heating region comprises a channel for fluid movement therethrough. In some embodiments, the channel is in fluid communication with the sample interface and the detection region, either directly or indirectly, thereby enabling the sample to move from the sample interface to the detection region. In some embodiments, the channel comprises a spiral configuration or a serpentine configuration. In some embodiments, two or more channels for fluid movement therethrough, wherein at least one channel of the two or more channels is configured to move the sample from the sample interface to the detection region. In some embodiments, each channel of the heating region comprises one or more movable mechanisms. In some embodiments, the one or more movable mechanisms comprises i) a first movable mechanism between the sample interface and heating region for controlling transfer of the sample therebetween, and/or ii) a second movable mechanism between the heating region and the detecting region for controlling transfer of the sample therebetween. In some embodiments, at least one channel of the heating region comprises two or more heating compartments configured to separate the sample into two or more sub-samples, wherein the two or more compartments are separated from each other via a movable mechanism of the one or more movable mechanisms. In some embodiments, each heating compartment is configured to be heated by a corresponding heating element. In some embodiments, at least one heating element of the device comprises a chemical heating element. In some embodiments, at least one chemical heating element is sodium acetate. In some embodiments, the heating region comprises a chamber in fluid communication with the sample interface and the detection region. In some embodiments, the heating region comprises a reporter immobilized therein, wherein the report is configured to release a detection moiety via the selective binding between the activated programmable nuclease and the target nucleic acid, thereby enabling the signal to be produced. In some embodiments, the reporter is immobilized in the heating region via a support that is immobilized on a surface of the heating region. In some embodiments, the support comprises a bead, a coating, and an interspersed polymer. In some embodiments, the support comprises a solid support. In some embodiments, the surface of the heating region comprises a well that is a recessed portion of the surface, wherein the support is disposed within the well. In some embodiments, one or more channels are configured to move the sample from the sample interface to the detection region. In some embodiments, the one or more channels are located within the sample interface, between the sample interface and the heating region, within the heating region, between the heating region and the detection region, and/or within the detection region. In some embodiments, one or more channels comprises a plurality of channels, wherein the plurality of channels comprises at least one set of channels arranged in series. In some embodiments, the one or more channels comprises a plurality of channels, wherein the plurality of channels comprises at least one set of channels arranged in parallel (parallel channels), thereby enabling the sample to be split into sub-samples within each channel of the at least one set of parallel channels. In some embodiments, the one or more channels comprises a plurality of channels, wherein the plurality of channels comprises at least one set of channels configured to move the sample from a first location within the device to a second location within the device, thereby enabling the sample to be split into sub-samples within each channel of the at least one set of channels In some embodiments, the at least one set of channels comprises two or more channels having a different length and/or different configuration, thereby enabling specific conditions to be specified for two or more corresponding sub-samples. In some embodiments, the specific conditions comprise a specified heating temperature range, a specified heating duration, a specified residence time within any region or location on the device, a specific incubation time, contact with specific reagents, or any combination thereof. In some embodiments, the at least one set of channels comprises two or more channels having a same length and/or configuration, thereby enabling specific conditions to be specified for two or more corresponding sub-samples. In some embodiments, a channel of the one or more channels comprises a radial configuration, a spiral configuration, a serpentine configuration, a linear configuration, or any combination thereof. In some embodiments, at least one actuator. In some embodiments, the at least one actuator comprises a plunger, a spring-actuated plunger, or a spring mechanism. In some embodiments, the at least one actuator is manually actuated. In some embodiments, a first actuator of the at least one actuator is configured to move the sample from the sample interface to the heating region via manual actuation of the first actuator. In some embodiments, a second actuator of the at least one actuator is configured to move the detection moiety from the heating region to the detection region via manual actuation of the second actuator. In some embodiments, the device is configured to be operated manually without electrical power. In some embodiments, a power source. In some embodiments, the power source comprises one or more batteries. In some embodiments, the heating region is configured to heat the sample via a heating element. In some embodiments, the heating element comprises a chemical heating element. In some embodiments, the chemical heating element is sodium acetate. In some embodiments, the signal is visually detectable. In some embodiments, the programmable nuclease comprises a guide nucleic acid. In some embodiments, the guide nucleic acid is modified. In some embodiments, the guide nucleic acid is modified with at least one methyl group. In some embodiments, the programmable nuclease further comprises a Cas enzyme. In some embodiments, the Cas enzyme is selected from the group consisting Cas12, Cas13, Cas14, Cas14a, Cas14a1, and CasPhi. In some embodiments, the target nucleic acid is indicative of a respiratory disorder or respiratory pathogen. In some embodiments, the respiratory disorder or respiratory pathogen selected from the group consisting of SARS-CoV-2 and corresponding variants, 29E), NL63, OC43, HKU1, MERS-CoV, (MERS), SARS-CoV (SARS, Flu A, Flu B, RSV, Rhinovirus, Strep A, and TB. In some embodiments, the device is configured to differentiate between a viral infection and a bacterial infection. In some embodiments, the target nucleic acid is indicative of a sexually transmitted infection (STI) or infection related to a woman's health. In some embodiments, the STI or infection related to a woman's health is selected from the group consisting of CT, NG, MG, TV, HPV, Candida, B. Vaginosis Syphilis and UTI. In some embodiments, the target nucleic acid comprises a single nucleotide polymorphism (SNP). In some embodiments, the SNP is indicative of NASH disorder or Alpha-1 disorder. In some embodiments, the target nucleic acid is a blood borne pathogen selected from the group consisted of HIV, HBV, HCV and Zika. In some embodiments, the target nucleic acid is indicative of H. Pylori, C. Difficile, Norovirus, HSV and Meningitis. In some embodiments, a physical filter configured to filter one or more particles from the sample that do not comprise the target nucleic acid. In some embodiments, the physical filter is located between and in fluid communication with the sample interface and heating region. In some embodiments, the programmable nuclease, guide nucleic acid, or the reporter are immobilized to a device surface by a linkage. In some embodiments, the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, a bond between streptavidin and biotin, an amide bond or any combination thereof. In some embodiments, the linkage comprises non-specific absorption. In some embodiments, the programmable nuclease is immobilized to the device surface by the linkage, wherein the linkage is between the programmable nuclease and the surface. In some embodiments, the reporter is immobilized to the device surface by the linkage, wherein the linkage is between the reporter and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 5′ end of the guide nucleic acid and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 3′ end of the guide nucleic acid and the surface. In some embodiments, a plurality of guide nucleic acids, wherein each guide nucleic acid of the plurality of guide nucleic acids is complementary, or partially complementary to a different segment of the target nucleic acid. In some embodiments, the sample comprises the sample containing the target nucleic acid(s), the sample containing the amplification reagents, the amplified sample, and/or the sample containing the detection moiety.
Described herein are various devices for detecting a target nucleic acid in a sample, comprising: a sample interface for receiving the sample; a reaction chamber (e.g., a heating region) in fluid communication with the sample interface, the heating region comprising: a programmable nuclease comprising a guide nucleic acid, and a reporter, wherein the programmable nuclease is activated by selective binding between the guide nucleic acid and a target nucleic acid, wherein the reporter is configured to release a detection moiety upon cleavage by the activated programmable nuclease; a chemical heating element configured to heat to the heating region; a detection region in fluid communication with the heating region, wherein the detection region is configured to detect a signal produced by the released detection moiety; a first manual actuator configured to transfer the sample from the heating region to the detection region; and a reagent mix comprising amplification reagents, wherein the reagent mix is disposed within the sample interface, the heating region, the detection region, and/or between the sample interface and the heating region, wherein the device is configured to determine the presence or absence of the target nucleic acid within a time of less than 30 minutes via the produced signal. In some embodiments, the reagent mix is lyophilized. In some embodiments, the heating region is configured to amplify the target nucleic acid. In some embodiments, the heating region comprises the amplification reagents. In some embodiments, the target nucleic acid is amplified via Loop-Mediated Isothermal Amplification (LAMP). In some embodiments, the heating region is configured to maintain an isothermal, or non-cycled temperature profile. In some embodiments, the isothermal, or non-cycled temperature profile is between about 30° C. to about 60° C. In some embodiments, the isothermal, or non-cycled temperature profile is about 55° C. to about 60° C. In some embodiments, the sample interface comprises a compartment configured to receive a swab containing the sample. In some embodiments, the compartment comprises a scraper configured to transfer the sample from the swab to the device. In some embodiments, the compartment contains an interface solution configured to extract the sample from the swab. In some embodiments, the interface solution comprises a buffer solution or a lysis buffer solution. In some embodiments, the sample interface is configured to receive the sample from a swab via pipetting. In some embodiments, the sample interface comprises a compartment configured to receive the sample from a container containing the sample. In some embodiments, the container comprises a syringe. In some embodiments, the syringe interface comprises an opening for receiving the sample therethrough. In some embodiments, the syringe interface opening is in fluid communication with the heating region, or another compartment that is in fluid communication with the heating region. In some embodiments, the sample interface is configured to receive the sample as a fluid. In some embodiments, the heating region and detection region are disposed on the same location on the device. In some embodiments, the heating region and the detection region are disposed within a same compartment of the device. In some embodiments, the heating region comprises a channel for fluid movement therethrough. In some embodiments, the channel is in fluid communication with the sample interface and the detection region, either directly or indirectly, thereby enabling the sample to move from the sample interface to the detection region. In some embodiments, the channel comprises a spiral configuration or a serpentine configuration. In some embodiments, two or more channels for fluid movement therethrough, wherein at least one channel of the two or more channels is configured to move the sample from the sample interface to the detection region. In some embodiments, each channel of the heating region comprises one or more movable mechanisms. In some embodiments, the one or more movable mechanisms comprises i) a first movable mechanism between the sample interface and heating region for controlling transfer of the sample therebetween, and/or ii) a second movable mechanism between the heating region and the detecting region for controlling transfer of the sample therebetween. In some embodiments, at least one channel of the heating region comprises two or more heating compartments configured to separate the sample into two or more sub-samples, wherein the two or more compartments are separated from each other via a movable mechanism of the one or more movable mechanism. In some embodiments, each heating compartment is configured to be heated by a corresponding heating element of at least one heating element. In some embodiments, the at least one heating element of the device comprises a chemical heating element. In some embodiments, the at least one chemical heating element is sodium acetate. In some embodiments, the heating region comprises a chamber. In some embodiments, the heating region comprises the reporter immobilized therein. In some embodiments, the reporter is immobilized in the heating region via a support that is immobilized on a surface of the heating region. In some embodiments, the support comprises a bead, a coating, and an interspersed polymer. In some embodiments, the support comprises a solid support. In some embodiments, the surface of the heating region comprises a well that is recessed portion of the surface, wherein the support is disposed within the well. In some embodiments, one or more channels are configured to move the sample from the sample interface to the detection region. In some embodiments, the one or more channels are located within the sample interface, between the sample interface and the heating region, within the heating region, between the heating region and the detection region, and/or within the detection region. In some embodiments, the one or more channels comprises a plurality of channels, wherein the plurality of channels comprises at least one set of channels arranged in series. In some embodiments, the one or more channels comprises a plurality of channels, wherein the plurality of channels comprises at least one set of parallel channels arranged in parallel (parallel channels), thereby enabling the sample to be split into sub-samples within each channel of the at least one set of parallel channels. In some embodiments, the one or more channels comprises a plurality of channels, wherein the plurality of channels comprises at least one set of channels configured to move the sample from a first location within the device to a second location within the device, thereby enabling the sample to be split into sub-samples within each channel of the at least one set of channels. In some embodiments, the at least one set of channels comprises two or more channels having a different length and/or different configuration, thereby enabling specific conditions to be specified for two or more corresponding sub-samples. In some embodiments, the specific conditions comprise a specified heating temperature range, a specified heating duration, a specified residence time within any region or location on the device, a specific incubation time, contact with specific reagents, or any combination thereof. In some embodiments, the at least one set of channels comprises two or more channels having a same length and/or configuration, thereby enabling specific conditions to be specified for two or more corresponding sub-samples. In some embodiments, a channel of the one or more channels comprises a radial configuration, a spiral configuration, a serpentine configuration, a linear configuration, or any combination thereof. In some embodiments, the at least one actuator comprises a plunger, a spring-actuated plunger, or a spring mechanism. In some embodiments, the device is configured to be operated manually without electrical power. In some embodiments, the device may comprise a power source. In some embodiments, the power source comprises one or more batteries. In some embodiments, the heating region is configured to heat the sample via a heating element. In some embodiments, the heating element comprises a chemical heating element. In some embodiments, the chemical heating element is sodium acetate. In some embodiments, the signal is visually detectable. In some embodiments, the guide nucleic acid is modified. In some embodiments, the guide nucleic acid is modified with at least one methyl group. In some embodiments, the programmable nuclease further comprises a Cas enzyme. In some embodiments, the Cas enzyme is selected from the group consisting Cas12, Cas13, Cas14, Cas14a, Cas14a1, and CasPhi. In some embodiments, the target nucleic acid is indicative of a respiratory disorder or respiratory pathogen. In some embodiments, the respiratory disorder or respiratory pathogen selected from the group consisting of SARS-CoV-2 and corresponding variants, 29E), NL63, OC43, HKU1, MERS-CoV, (MERS), SARS-CoV (SARS, Flu A, Flu B, RSV, Rhinovirus, Strep A, and TB. In some embodiments, the device is configured to differentiate between a viral infection and a bacterial infection. In some embodiments, the target nucleic acid is indicative of a sexually transmitted infection (STI) or infection related to a woman's health. In some embodiments, the STI or infection related to a woman's health is selected from the group consisting of CT, NG, MG, TV, HPV, Candida, B. Vaginosis Syphilis, and UTI. In some embodiments, the target nucleic acid comprises a single nucleotide polymorphism (SNP). In some embodiments, the SNP is indicative of NASH disorder or Alpha-1 disorder. In some embodiments, the target nucleic acid is a blood borne pathogen selected from the group consisted of HIV, HBV, HCV, and Zika. In some embodiments, the target nucleic acid is indicative of H. Pylori, C. Difficile, Norovirus, HSV, and Meningitis. In some embodiments, a physical filter configured to filter one or more particles from the sample that do not comprise the target nucleic acid. In some embodiments, the physical filter is located between and in fluid communication with the sample interface and heating region. In some embodiments, the programmable nuclease, guide nucleic acid, or the reporter are immobilized to a device surface by a linkage. In some embodiments, the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, a bond between streptavidin and biotin, an amide bond or any combination thereof. In some embodiments, the linkage comprises non-specific absorption. In some embodiments, the programmable nuclease is immobilized to the device surface by the linkage, wherein the linkage is between the programmable nuclease and the surface. In some embodiments, the reporter is immobilized to the device surface by the linkage, wherein the linkage is between the reporter and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 5′ end of the guide nucleic acid and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 3′ end of the guide nucleic acid and the surface. In some embodiments, a plurality of guide nucleic acids, wherein each guide nucleic acid of the plurality of guide nucleic acids is complementary, or partially complementary to a different segment of the target nucleic acid. In some embodiments, the sample comprises the sample containing the target nucleic acid(s), the sample containing the amplification reagents, the amplified sample, and/or the sample containing the detection moiety.
Described herein are various embodiments for a microarray device for multiplexed detection of a plurality of target nucleic acids in a sample, comprising: a sample interface for receiving the sample; a reagent mix comprising amplification reagents; a reaction chamber (e.g., a heating region) in fluid communication with the sample interface; and a detection region comprising a surface comprising a plurality of detection spots in a microarray format, wherein each of the plurality of detection spots comprise a reporter probe, wherein each reporter probe is configured to release a detection moiety via cleavage by an activated programmable nuclease, wherein each of the plurality of detection spots comprise a different programmable nuclease probe, and wherein the device is configured to determine the presence or absence of each of the plurality of target nucleic acids within a time of less than 30 minutes via the release of each detection moiety of each reporter at each of the plurality of detection spots. In some embodiments, at least one programmable nuclease probe comprises a Cas enzyme. In some embodiments, each Cas enzyme of the at least one programmable nuclease is selected from the group comprising of Cas12, Cas13, Cas14, Cas14a, and Cas14a1. In some embodiments, the microarray device may comprise all the various embodiments of devices as described herein.
Described herein are various embodiment of a kit for the detection of a target nucleic acid in a sample, the kit comprising: a swab; elution reagents; lysis reagents; a device comprising a sample interface for receiving the sample; a reaction chamber (e.g., a heating region) in fluidic communication with the sample interface and configured to receive the sample, the heating region comprising a programmable nuclease comprising a guide nucleic acid and a reporter disposed within the heating region, wherein the programmable nuclease is activated by selective binding between the guide nucleic acid and the target nucleic acid, wherein the reporter is configured to release a detection moiety via the activated programmable nuclease; a chemical heating element configured to provide heat to the heating region; a detection region in fluid communication with the heating region and the sample interface, wherein the detection region is configured to detect a signal produced by the released detection moiety, thereby detecting the presence of the target nucleic acid; and a reagent mix comprising amplification reagents, wherein the reagent mix is disposed within the sample interface, the heating region, the detection region, and/or between the sample interface and the heating region, and wherein the device is configured to determine the presence or absence of the target nucleic acid within a time of less than 30 minutes via the released detection moiety. In some embodiments, the sample interface is configured to receive the sample from the swab. In some embodiments, a collection tube, wherein the collection tube is configured to accept the swab, wherein the sample contained in the swab is transferred to the collection tube, and wherein the collection tube is separate from the device. In some embodiments, the collection tube is configured to be inserted into the sample interface to transfer the sample to the device. In some embodiments, the collection tube is a syringe. In some embodiments, the kit comprises a first container containing the elution reagents and/or the lysis reagents. In some embodiments, the kit comprises dilution reagents. In some embodiments, the kit comprises a second container containing the dilution reagents. In some embodiments, the programmable nuclease comprises a Cas enzyme. In some embodiments, the Cas enzyme is selected from the group comprising of Cas12, Cas13, Cas14, Cas14a, and Cas14a1. In some embodiments, the kit may comprise all the various embodiments of devices, as described herein.
Described herein are various embodiments of a method for the detection of a target nucleic acid in a sample, the method comprising: providing a device configured to determine a presence or absence of a target nucleic acid in less than 30 minutes after a sample is introduced into the device, the device comprising a sample interface, a heating region in fluid communication with the sample interface, and a detection region in fluid communication with the heating region; introducing the sample into the sample interface of the device; mixing the sample with a reagent mix comprising amplification reagents to generate a mixed sample solution; transferring the mixed sample solution from the sample interface to the heating region; amplifying the sample by heating the mixed sample solution in the heating region; performing a programmable nuclease-based assay, wherein selective binding between a guide nucleic acid and the target nucleic acid activates a programmable nuclease probe configured to cleave a reporter probe, thereby releasing a detection moiety into the sample solution when the target nucleic acid is present; transferring the mixed sample solution with the amplified sample from the heating region to the detection region; and determining the presence or absence of the target nucleic acid in the sample via capture of the released detection moiety in the detection region. In some embodiments, the amplifying the target nucleic acid and the performing the programmable nuclease assay are performed as a one-pot reaction in the heating region. In some embodiments, the one-pot reaction is performed between about 30° C. to 60° C. In some embodiments, the one-pot reaction is performed at about 55° C. In some embodiments, the programmable nuclease of the one-pot reaction comprises a Cas enzyme, the Cas enzyme selected from the group comprising of Cas12, Cas13, Cas14, Cas14a, and Cas14a1. In some embodiments, the method further comprises filtering the sample with the reagent mix prior to entering the heating region. In some embodiments, the method further comprises filtering the sample comprises filtering one or more particles from the sample that do not comprise the target nucleic acid. In some embodiments, the filter is located between and in fluid communication with the sample interface and heating region. In some embodiments, the method further comprises a plurality of guide nucleic acids, each guide nucleic acid of the plurality of guide nucleic acids is complementary, or partially complementary to a different segment of the target nucleic acid. In some embodiments, the method further comprises, prior to step (a): providing a collection tube comprising a sample solution comprising the target nucleic acid; and transferring the sample solution to the device via inserting the collection tube into a sample interface of the device, wherein the sample solution dissolves and mixes with a lyophilized reaction mix comprising amplification reagents. In some embodiments, the method may comprise all of the embodiments the various devices, as described herein.
Described herein are various devices for detecting a target nucleic acid, comprising: a sample interface configured to receive a sample comprising the target nucleic acid; a first reaction chamber in fluid communication with the sample interface, wherein the first reaction chamber comprises a plurality of reporters; and a second reaction chamber in fluid communication with the first reaction chamber, wherein the second reaction chamber comprises an enzyme substrate, wherein each reporter of the plurality of reporters (i) is immobilized to a surface of the reaction chamber via a tether, (ii) comprised of a programmable nuclease-enzyme fusion protein complexed with a guide nucleic acid, the guide nucleic acid being configured to bind to the target nucleic acid, and (iii) configured to release the programmable nuclease-enzyme fusion protein following cleavage of the tether by the programmable nuclease-enzyme fusion protein bound to the target nucleic acid, and wherein release of the programmable nuclease-enzyme fusion protein is indicative of a presence or absence of the target nucleic acid in the sample. In some embodiments, the programmable nuclease-enzyme fusion protein comprises Cas12-HRP. In some embodiments, the tether of each reporter of the plurality of reporters comprises ssDNA. In some embodiments, the tether of each reporter of the plurality of reporters comprises RNA. In some embodiments, the tether of each reporter of the plurality of reporters comprises both ssDNA and RNA. In some embodiments, the programmable nuclease-enzyme fusion protein comprises Cas12-HRP. In some embodiments, the tether of each reporter of the plurality of reporters comprises RNA. In some embodiments, the enzyme substrate is HRP substrate. In some embodiments, the surface is configured to immobilize a nucleic acid. In some embodiments, the surface comprises streptavidin, biotin, an amine group, a carboxyl group, an epoxy group, an NHS group, a malemide group, or a thiol group. In some embodiments, the surface is a hydrogel surface. In some embodiments, the surface comprises an acrydite modification.
Described herein are various devices for detecting a target nucleic acid, comprising: a sample interface configured to receive a sample comprising the target nucleic acid; a first reaction chamber in fluid communication with the sample interface, wherein the first reaction chamber comprises a plurality of reporters; a second reaction chamber in fluid communication with the first reaction chamber, wherein the second reaction chamber comprises an enzyme substrate, wherein each reporter of the plurality of reporters comprises a programmable nuclease-split enzyme fusion protein complexed with a guide nucleic acid, the guide nucleic acid being configured to bind to the target nucleic acid, and wherein release of the programmable nuclease-split enzyme fusion protein is indicative of a presence or absence of the target nucleic acid in the sample. In some embodiments, the programmable nuclease-split enzyme fusion protein comprises HRP-L. In some embodiments, the device further comprises a programmable nuclease-split enzyme fusion protein comprising HRP-S. In some embodiments, the device is configured to produce a signal upon binding of the programmable nuclease-split enzyme fusion protein comprising HRP-L and binding of the programmable nuclease-split enzyme fusion protein comprising HRP-S to the target nucleic acid.
Described herein are various devices for detecting a target nucleic acid, comprising: a sample interface configured to receive a sample comprising the target nucleic acid; a first reaction chamber in fluid communication with the sample interface, wherein the first reaction chamber comprises a plurality of reporters, each reporter of the plurality comprising two or more [enzymes]; and a surface of the first reaction chamber, wherein each reporter of the plurality of reporters is immobilized to the surface of the first reaction chamber via a tether, and a second reaction chamber in fluid communication with the first reaction chamber, wherein the second reaction chamber comprises an enzyme substrate, and wherein cleaving the tether by a programmable nuclease complexed with a guide nucleic acid, the guide nucleic acid being configured to bind to the target nucleic acid, may release the two or more enzymes into solution, and wherein each enzyme of the two or more enzymes may be contacted with the enzyme substrate in the second reaction chamber to create a detectable and amplified signal indicative of a presence or absence of the target nucleic acid in the sample. In some embodiments, each enzyme of the two or more enzymes comprises HRP. In some embodiments, the reporter comprises a streptavidin comprising the two or more enzymes. In some embodiments, the tether contacts the surface via a biotin, an acrydite, or an amine. In some embodiments, the surface of the first reaction chamber is functionalized with a hydrogel, carboxyl group, or N-Hydroxysuccinimide (NHS). In some embodiments, the [tether] comprises a segment of cleavable DNA, cleavable RNA or a combination thereof. In some embodiments, the enzyme substrate comprises 3,3′,5,5′-tetramethylbenzidine (TMB). In some embodiments, the surface of the first reaction chamber comprises an inner wall of the first reaction chamber. In some embodiments, the surface of the first reaction chamber comprises a surface of a bead, wherein the bead is contained within the first reaction chamber. In some embodiments, the bead is a magnetic bead.
In one aspect, the present disclosure provides a composition for detecting a target nucleic acid, the composition comprising a programmable nuclease, a guide nucleic acid, a first reporter, an enzyme, and a second reporter. In some embodiments, (a) the guide nucleic acid is configured to bind to the target nucleic acid; (b) the programmable nuclease is effective to cleave the first reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (c) the cleavage of the first reporter is effective to separate a first nucleic acid section from a second nucleic acid section thereof; (d) the first nucleic acid section is effective to activate the enzyme; and (e) the activated enzyme is effective to cleave the second reporter to produce a detectable product comprising a detection moiety. In some embodiments, the enzyme is an endonuclease (e.g., NucC) and the second reporter comprises a polynucleotide substrate of the enzyme (e.g., cyclic triadenylate (cA3)). In some embodiments, the second nucleic acid section comprises RNA residues, optionally wherein the RNA residues comprise a plurality of uracil residues. In some embodiments, the second nucleic acid section comprises DNA residues, optionally wherein the DNA residues comprise a plurality of thymine residues. In some embodiments, (a) the second reporter comprises a fluorescent label and a quencher, and (b) cleavage of the second reporter by the activated enzyme is effective to separate the fluorescent label from the quencher.
In one aspect, the present disclosure provides a method of detecting a target nucleic acid in a sample. In some embodiments, the method comprises (a) contacting the sample with a composition described herein (e.g., a composition comprising a programmable nuclease, a guide nucleic acid, a first reporter, an enzyme, and a second reporter); (b) cleaving the first reporter with the programmable nuclease in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid, thereby releasing the first nucleic acid section; (c) activating the enzyme with the first nucleic acid section; (d) cleaving the second reporter with the activated enzyme, thereby producing the detectable product comprising the detection moiety; and (e) detecting the detection moiety, thereby detecting the presence of the target nucleic acid in the sample. In some embodiments, the second reporter comprises a polynucleotide substrate of the enzyme, and the enzyme is a NucC. In some embodiments, step (d) is performed at a temperature of at least 40° C. In some embodiments, all of steps (b)-(d) are performed at a temperature of at least 40° C. (e.g., 45° C., 50° C., 55° C., 60° C., 65° C., or higher).
In one aspect, the present disclosure provides a composition for detecting a target nucleic acid in a reaction chamber, the composition comprising a programmable nuclease, a guide nucleic acid, a forward primer, a reverse primer, a polymerase, a nicking endonuclease, and a reporter. In some embodiments, (a) the guide nucleic acid is configured to bind to the target nucleic acid; (b) the forward primer comprises (i) a 5′ portion comprising a first hairpin, and (ii) a 3′ portion that is configured to bind the target nucleic acid at a first overlapping region with respect to the guide nucleic acid; (c) the reverse primer comprises (i) a 5′ portion comprising a second hairpin, and (ii) a 3′ portion that is configured to bind a complement of the target nucleic acid at a second overlapping region with respect to the guide nucleic acid; (d) the first and second hairpins are cleavage substrates for the nicking endonuclease; (e) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and (i) the target nucleic acid or (ii) an amplicon of the target nucleic acid; and (f) the cleavage of the reporter is effective to produce a detectable product comprising a detection moiety. In some embodiments, (a) the sequence of the target nucleic acid to which the 3′ portion of the first primer is configured to bind defines a first sequence of the target nucleic acid; (b) the sequence of the 3′ portion of the reverse primer defines a second sequence of the target nucleic acid; and (c) the first sequence and second sequence are separated by about 5 to about 10 nucleotides along the target nucleic acid. In some embodiments, the 3′ portions of the forward primer and reverse primer are about 16 to about 20 nucleotides in length. In some embodiments, overlap between the 3′ portion of the reverse primer and the sequence to which the guide nucleic acid is configured to bind overlap by 1 to 5 nucleotides, 2 to 5 nucleotides, or 3 nucleotides. In some embodiments, the first hairpin and/or the second hairpin are 10 to 20 nucleotides in length, 16 to 20 nucleotides in length, or 16 nucleotides in length. In some embodiments, the programmable nuclease is a Cas protein, optionally wherein the Cas protein is a Cas12 protein or a Cas14 protein.
In a related aspect, the present disclosure provides a method of detecting a target nucleic acid in a sample using a composition described herein (e.g., a composition comprising a programmable nuclease, a guide nucleic acid, a forward primer, a reverse primer, a polymerase, a nicking endonuclease, and a reporter). In some embodiments, the method comprises: (a) contacting the sample with the composition; (b) performing nicking enzyme amplification reaction (NEAR) reaction to amplify the target nucleic acid; (c) forming a complex comprising the programmable nuclease, the guide nucleic acid, and (i) the target nucleic acid, or (ii) an amplicon of the target nucleic acid; (d) cleaving the reporter with the programmable nuclease activated by formation of the complex, thereby producing the detectable cleavage product; and (e) detecting the detection moiety, thereby detecting the presence of the target nucleic acid in the sample. In some embodiments, steps (b) through (d) are performed at about the same temperature (e.g., as in an isothermal reaction).
In one aspect, the present disclosure provides systems comprising a reaction chamber comprising a composition described herein, such as with regard to any of the various aspects described herein. In some embodiments, the reaction chamber is a chamber within a device disclosed herein.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
(Cas12 variant DETECTR using SEQ ID NO: 17) control run.
The present disclosure provides systems and methods for nucleic acid target detection. The systems and methods of the present disclosure can be implemented using devices that are configured for programmable nuclease-based detection. In some embodiments, the devices can be configured for single reaction detection. In some embodiments, the devices can be disposable devices. The devices disclosed herein can be particularly well suited for carrying out highly efficient, rapid, and accurate reactions for detecting whether a target is present in a sample. The target can comprise a target sequence or target nucleic acid. As used herein, a target can be referred to interchangeably as a target nucleic acid. Further, a target can be referred to as a target amplicon or a target nucleic acid amplicon if such target undergoes amplification (e.g., through a thermocycling process as described elsewhere herein). The target nucleic acid can be a portion of a nucleic acid of interest, e.g., a target nucleic acid from any plant, animal, virus, or microbe of interest. The devices provided herein can be used to perform rapid tests in a single integrated system.
The target nucleic acid can be a nucleic acid or a portion of a nucleic acid from a pathogen, virus, bacterium, fungi, protozoa, worm, or other agent(s) or organism(s) responsible for and/or related to a disease or condition in living organisms (e.g., humans, animals, plants, crops, and the like). The target nucleic acid can be a nucleic acid, or a portion thereof. The target nucleic acid can be a portion of a nucleic acid from a gene expressed in a cancer or genetic disorder in the sample. The target nucleic acid can be a portion of an RNA or DNA from any organism in the sample. In some embodiments, one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule). A programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter. The programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter. Such a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein can be activated by a target RNA or a target DNA. For example, a programmable nuclease, e.g., a Cas enzyme, can be activated by a target RNA nucleic acid or a target DNA nucleic acid to cleave RNA reporters. In some embodiments, the Cas enzyme can bind to a target ssDNA which initiates trans cleavage of RNA reporters. In some instances, a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA-activated programmable DNA nuclease.
The nucleic acids described and referred to herein can comprise a plurality of base pairs. A base pair can be a biological unit comprising two nucleobases bound to each other by hydrogen bonds. Nucleobases can comprise adenine, guanine, cytosine, thymine, and/or uracil. In some cases, the nucleic acids described and referred to herein can comprise different base pairs. In some cases, the nucleic acids described and referred to herein can comprise one or more modified base pairs. The one or more modified base pairs can be produced when one or more base pairs undergo a chemical modification leading to new bases. The one or more modified base pairs can be, for example, Hypoxanthine, Inosine, Xanthine, Xanthosine, 7-Methylguanine, 7-Methylguanosine, 5,6-Dihydrouracil, Dihydrouridine, 5-Methylcytosine, 5-Methylcytidine, 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC).
The programmable nuclease can become activated after binding of a guide nucleic acid that is complexed with the programmable nuclease with a target nucleic acid, and the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the target nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal. The reporter and/or the detection moiety can be immobilized on a support medium. Often the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid. 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 concentration of one or more target nucleic acids associated with an ailment, such as a disease, cancer, or genetic disorder.
The systems and methods of the present disclosure can be implemented using a device that is compatible with any type of programmable nuclease that is human-engineered or naturally occurring. The programmable nuclease can comprise a nuclease that is capable of being activated when complexed with a guide nucleic acid and a target nucleic acid segment or a portion thereof. A programmable nuclease can become activated when complexed with a guide nucleic acid and a target sequence of a target gene of interest. The programmable nuclease can be activated upon binding of a guide nucleic acid to a target nucleic acid and can exhibit or enable trans cleavage activity once activated. In any instances or embodiments where a CRISPR-based programmable nuclease is described or used, it is recognized herein that any other type of programmable nuclease can be used in addition to or in substitution of such CRISPR-based programmable nuclease.
The systems and methods of the present disclosure can be implemented using a device that is compatible with a plurality of programmable nucleases. The device can comprise a plurality of programmable nuclease probes comprising the plurality of programmable nucleases and one or more corresponding guide nucleic acids. The plurality of programmable nuclease probes can be the same. Alternatively, the plurality of programmable nuclease probes can be different. For example, the plurality of programmable nuclease probes can comprise different programmable nucleases and/or different guide nucleic acids associated with the programmable nucleases.
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 Cpf1. Programmable nucleases can also include, for example, PfAgo and/or NgAgo.
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.
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.
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.
Disclosed herein are programmable nucleases and uses thereof, e.g., detection and editing of target nucleic acids. In some instances, programmable nucleases comprise a Type V CRISPR/Cas protein. In some instances, Type V CRISPR/Cas proteins comprise nucleic acid cleavage activity. In some instances, Type V CRISPR/Cas proteins cleave or nick single-stranded nucleic acids, double, stranded nucleic acids, or a combination thereof. In some cases, Type V CRISPR/Cas proteins cleave single-stranded nucleic acids. In some cases, Type V CRISPR/Cas proteins cleave double-stranded nucleic acids. In some cases, Type V CRISPR/Cas proteins nick double-stranded nucleic acids. Typically, guide RNAs of Type V CRISPR/Cas proteins hybridize to ssDNA or dsDNA. However, the trans cleavage activity of Type V CRISPR/Cas protein is typically directed towards ssDNA. In some cases, the Type V CRISPR/Cas protein comprises a catalytically inactive nuclease domain. In some cases, the Type V CRISPR/Cas protein comprises a catalytically inactive nuclease domain. A catalytically inactive domain of a Type V CRISPR/Cas protein may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to a wild type nuclease domain of the Type V CRISPR/Cas protein. Said mutations may be present within a cleaving or active site of the nuclease. The Type V CRISPR/Cas protein may be a Cas14 protein. The Cas 14 protein may be a Cas14a. 1 protein. The Cas14a. 1 protein may be represented by SEQ ID NO: 3, presented in Table 1. The Cas14 protein may comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3. The Cas14 protein may consist of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3. The Cas14 protein may comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of SEQ ID NO: 3.
Table 1 provides illustrative amino acid sequences of programmable nucleases having trans-cleavage activity. In some instances, programmable nucleases described herein comprise an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID Nos: 1-34 or 87-125. The programmable nuclease may consist of an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one or SEQ ID Nos: 1-34 or 87-125. The programmable nuclease may comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of any one of SEQ ID NOs: 1-34 or 87-125.
Other Exemplary protein sequences are described in the following applications: PCT/US2021/033271; PCT/US2021/035031, and PCT/US2022/028865, all of which are herein incorporated by reference in their entirety.
In some instances, the Type V CRISPR/Cas protein has been modified (also referred to as an engineered protein). For example, a Type V CRISPR/Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS). In some cases, the NLS may comprise a sequence of KRPAATKKAGQAKKKKEF (SEQ ID NO: 128). Type V CRISPR/Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the Type V CRISPR/Cas protein is codon optimized for a human cell.
In some instances, the Type V CRISPR/Cas protein has been modified (also referred to as an engineered protein). For example, a Type V CRISPR/Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS). In some cases, the NLS may comprise a sequence of KRPAATKKAGQAKKKKEF (SEQ ID NO: 128). Type V CRISPR/Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the Type V CRISPR/Cas protein is codon optimized for a human cell.
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.
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.
Cas 14 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 Cas 14 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 Cas 14 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 Cas 14 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.
Cas 14 proteins may comprise a zinc finger domain. In some instances, a carboxy terminal domain of a Cas 14 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.
Cas 14 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.
In some instances, a Cas 14 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 Cas 14 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 detector nucleic acids with a detection moiety.
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.
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.
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.
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.
Described herein are various embodiments of programmable nuclease fusion proteins. In some embodiments, a programmable nuclease may be fused with a heterologous polypeptide to form a programmable nuclease fusion protein. In some instances, “fusion partner” can refer to a polypeptide that is fused to another polypeptide. In some embodiments, the heterologous polypeptide may be an enzyme, wherein the enzyme is configured to react an enzyme substrate to generate a detectable signal. In some embodiments, the heterologous polypeptide may provide a tag (i.e., the heterologous polypeptide is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a histidine tag, e.g., a 6×His tag (SEQ ID NO: 129); a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).
“Heterologous,” as used herein, can refer to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. In some cases, a heterologous polypeptide may comprise a sequence different from a sequence of a given protein. For example, a sequence of an HRP enzyme may be considered heterologous to a sequence of a programmable nuclease when the sequence of the HRP enzyme is different from the sequence of the programmable nuclease. In some embodiments, a programmable nuclease (e.g., a Cas12 protein) can be fused to an active domain from a non-programmable nuclease protein (e.g., an HRP), and the sequence of the active domain may be considered to be a heterologous polypeptide (i.e., it is heterologous to the programmable nuclease).
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.
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.
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.
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.
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.
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.
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.
The reporters described herein can be RNA reporters. The RNA reporters can comprise at least one ribonucleic acid and a detectable moiety. In some embodiments, a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect ssDNA and, further, can specifically trans-cleave RNA reporters. The detection of the target nucleic acid in the sample can indicate the presence of the disease (or disease-causing agent) in the sample and can provide information for taking action to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual.
Cleavage of a reporter (e.g., a protein-nucleic acid) can produce a signal. The signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample. In some cases, cleavage of the protein-nucleic acid can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample. The sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.
In an aspect, the present disclosure provides a method for target detection. The method can comprise sample collection. The method can further comprise sample preparation. The method can further comprise detection of one or more target nucleic acids in the collected and prepared sample.
In another aspect, the present disclosure provides a detection device for target detection. The detection device can be configured for multiplexed target detection. The detection device can be used to collect one or more samples, prepare or process the one or more samples for detection, and optionally divide the one or more samples into a plurality of droplets, aliquots, or subsamples for amplification of one or more target sequences or target nucleic acids. The target sequences may comprise, for example, a biological sequence. The biological sequence can comprise a nucleic acid sequence or an amino acid sequence. In some embodiments, the target sequences are associated with an organism of interest, a disease of interest, a disease state of interest, a phenotype of interest, a genotype of interest, or a gene of interest.
The detection device can be configured to amplify target nucleic acids contained within the plurality of droplets, aliquots, or subsamples. The detection device can be configured to amplify the target sequences or target nucleic acids contained within the plurality of droplets by individually processing each of the plurality of droplets (e.g., by using a thermocycling process or any other suitable amplification process as described in greater detail below). In some cases, the plurality of droplets can undergo separate thermocycling processes. In some cases, the thermocycling processes can occur simultaneously. In other cases, the thermocycling processes can occur at different times for each droplet.
The detection device can be further configured to remix the droplets, aliquots, or subsamples after the target nucleic acids in each of the droplets undergo amplification. The detection device can be configured to provide the remixed sample comprising the droplets, aliquots, or subsamples to a detection chamber of the device. The detection chamber can be configured to direct the remixed droplets, aliquots, or subsamples to a plurality of programmable nuclease probes. The detection chamber can be configured to direct the remixed droplets, aliquots, or subsamples along one or more fluid flow paths such that the remixed droplets, aliquots, or subsamples are positioned adjacent to and/or in contact with the one or more programmable nuclease probes. In some cases, the detection chamber can be configured to recirculate or recycle the remixed droplets, aliquots, or subsamples such that the remixed droplets, aliquots, or subsamples are repeatedly placed in contact with one or more programmable nuclease probes over a predetermined period of time.
The detection device can comprise one or more sensors. The one or more sensors of the detection device can be configured to detect one or more signals that are generated after one or more programmable nucleases of the one or more programmable nuclease probes become activated due to a binding of a guide nucleic acid of the programmable nuclease probes with a target nucleic acid present in the sample. As described elsewhere herein, the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity. Trans cleavage activity can be a non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of target nucleic acids with a detection moiety. Once the target nucleic acids are cleaved by the activated programmable nucleases, the detection moiety can be released or separated from the reporter, thereby generating one or more detectable signals. The one or more sensors of the detection device can be configured to register and/or process the one or more detectable signals to confirm a presence and/or an absence of a particular target (e.g., a target nucleic acid).
The one or more programmable nuclease probes of the detection device can be configured for multiplexed detection. In some cases, each programmable nuclease probe can be configured to detect a particular target. In other cases, each programmable nuclease probe can be configured to detect a plurality of targets. In some cases, a first programmable nuclease probe can be configured to detect a first target or a first set of targets, and a second programmable nuclease probe can be configured to detect a second target or a second set of targets. In other cases, a first programmable nuclease probe can be configured to detect a first set of targets, and a second programmable nuclease probe can be configured to detect a second set of targets. The programmable nuclease probes of the present disclosure can be used to detect a plurality of different target sequences or target nucleic acids. In any of the embodiments described herein, the sample provided to the detection device can comprise a plurality of target sequences or target nucleic acids. In any of the embodiments described herein, the sample provided to the detection device can comprise multiple classes of target sequences or target nucleic acids. Each class of target sequences or class of target nucleic acids can comprise a plurality of target sequences or target nucleic acids associated with a particular organism, disease state, phenotype, or genotype present within the sample. In some cases, each programmable nuclease probe can be used to detect a particular class of target sequences or a particular class of target nucleic acids associated with a particular organism, disease state, phenotype, or genotype present within the sample. In some cases, two or more programmable nuclease probes can be used to detect different classes of target sequences or different classes of target nucleic acids. In such cases, the two or more programmable nuclease probes can comprise different sets or classes of guide nucleic acids complexed to the programmable nucleases of the probes.
In any of the embodiments described herein, the detection device can comprise a single integrated system that is configured to perform sample collection, sample processing, droplet generation, droplet processing (e.g., amplification of target nucleic acids in droplets), droplet remixing, and/or circulation of the remixed droplets within a detection chamber so that at least a portion of the remixed droplets is placed in contact with one or more programmable nuclease probes coupled to the detection chamber. The detection devices of the present disclosure can be disposable devices configured to perform one or more rapid single reaction or multi-reaction tests to detect a presence and/or an absence of one or more target sequences or target nucleic acids.
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.
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.
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.
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.
In some embodiments, the Coronavirus HKU1 sequence is a target of an assay. In some embodiments, the Coronavirus NL63 sequence is a target of an assay. In some embodiments, the Coronavirus 229E sequence is a target of an assay. In some embodiments, the Coronavirus OC43 sequence is a target of an assay. In some embodiments, the SARS-CoV-1 sequence is a target of an assay. In some embodiments, the MERS sequence is a target of an assay. In some embodiments, the SARS-CoV-2 sequence is a target of an assay. In some embodiments, the Respiratory Syncytial Virus A sequence is a target of an assay. In some embodiments, the Respiratory Syncytial Virus B sequence is a target of an assay. In some embodiments, the Influenza A sequence is a target of an assay. In some embodiments, the Influenza B sequence is a target of an assay. In some embodiments, the Human Metapneumovirus sequence is a target of an assay. In some embodiments, the Human Rhinovirus sequence is a target of an assay. In some embodiments, the Human Enterovirus sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 1 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 2 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 3 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 4 sequence is a target of an assay. In some embodiments, the Alphacoronavirus genus sequence is a target of an assay. In some embodiments, the Betacoronavirus genus sequence is a target of an assay. In some embodiments, the Sarbecovirus subgenus sequence is a target of an assay. In some embodiments, the SARS-related virus species sequence is a target of an assay. In some embodiments, the Gammacoronavirus Genus sequence is a target of an assay. In some embodiments, the Deltacoronavirus Genus sequence is a target of an assay. In some embodiments, the Influenza B-Victoria V1 sequence is a target of an assay. In some embodiments, the Influenza B-Yamagata Y1 sequence is a target of an assay. In some embodiments, the Influenza A H1 sequence is a target of an assay. In some embodiments, the Influenza A H2 sequence is a target of an assay. In some embodiments, the Influenza A H3 sequence is a target of an assay. In some embodiments, the Influenza A H4 sequence is a target of an assay. In some embodiments, the Influenza A H5 sequence is a target of an assay. In some embodiments, the Influenza A H6 sequence is a target of an assay. In some embodiments, the Influenza A H7 sequence is a target of an assay. In some embodiments, the Influenza A H8 sequence is a target of an assay. In some embodiments, the Influenza A H9 sequence is a target of an assay. In some embodiments, the Influenza A H10 sequence is a target of an assay. In some embodiments, the Influenza A H11 sequence is a target of an assay. In some embodiments, the Influenza A H12 sequence is a target of an assay. In some embodiments, the Influenza A H13 sequence is a target of an assay. In some embodiments, the Influenza A H14 sequence is a target of an assay. In some embodiments, the Influenza A H15 sequence is a target of an assay. In some embodiments, the Influenza A H16 sequence is a target of an assay. In some embodiments, the Influenza A N1 sequence is a target of an assay. In some embodiments, the Influenza A N2 sequence is a target of an assay. In some embodiments, the Influenza A N3 sequence is a target of an assay. In some embodiments, the Influenza A N4 sequence is a target of an assay. In some embodiments, the Influenza A N5 sequence is a target of an assay. In some embodiments, the Influenza A N6 sequence is a target of an assay. In some embodiments, the Influenza A N7 sequence is a target of an assay. In some embodiments, the Influenza A N8 sequence is a target of an assay. In some embodiments, the Influenza A N9 sequence is a target of an assay. In some embodiments, the Influenza A N10 sequence is a target of an assay. In some embodiments, the Influenza A N11 sequence is a target of an assay. In some embodiments, the Influenza A/H1-2009 sequence is a target of an assay. In some embodiments, the Human endogenous control 18S rRNA sequence is a target of an assay. In some embodiments, the Human endogenous control GAPDH sequence is a target of an assay. In some embodiments, the Human endogenous control HPRT1 sequence is a target of an assay. In some embodiments, the Human endogenous control GUSB sequence is a target of an assay. In some embodiments, the Human endogenous control RNASe P sequence is a target of an assay. In some embodiments, the Influenza A oseltamivir resistance sequence is a target of an assay. In some embodiments, the Human Bocavirus sequence is a target of an assay. In some embodiments, the SARS-CoV-2 85Δ sequence is a target of an assay. In some embodiments, the SARS-CoV-2 T1001I sequence is a target of an assay. In some embodiments, the SARS-CoV-2 3675-36774 sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P4715L sequence is a target of an assay. In some embodiments, the SARS-CoV-2 S5360L sequence is a target of an assay. In some embodiments, the SARS-CoV-2 69-70Δ sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Tyr144fs sequence is a target of an assay. In some embodiments, the SARS-CoV-2 242-244Δ sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Y453F sequence is a target of an assay. In some embodiments, the SARS-CoV-2 S477N sequence is a target of an assay. In some embodiments, the SARS-CoV-2 E848K sequence is a target of an assay. In some embodiments, the SARS-CoV-2 N501Y sequence is a target of an assay. In some embodiments, the SARS-CoV-2 D614G sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P681R sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P681H sequence is a target of an assay. In some embodiments, the SARS-CoV-2 L21F sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Q27Stop sequence is a target of an assay. In some embodiments, the SARS-CoV-2 M1fs sequence is a target of an assay. In some embodiments, the SARS-CoV-2 R203fs sequence is a target of an assay. In some embodiments, the Human adenovirus-pan assay sequence is a target of an assay. In some embodiments, the Bordetella parapertussis sequence is a target of an assay. In some embodiments, the Bordetella pertussis sequence is a target of an assay. In some embodiments, the Chlamydophila pneumoniae sequence is a target of an assay. In some embodiments, the Mycoplasma pneumoniae sequence is a target of an assay. In some embodiments, the Legionella pneumophila sequence is a target of an assay. In some embodiments, the Bordetella bronchoseptica sequence is a target of an assay. In some embodiments, the Bordetella holmesii sequence is a target of an assay. In some embodiments, the Human adenovirus Type A sequence is a target of an assay. In some embodiments, the Human adenovirus Type B sequence is a target of an assay. In some embodiments, the Human adenovirus Type C sequence is a target of an assay. In some embodiments, the Human adenovirus Type D sequence is a target of an assay. In some embodiments, the Human adenovirus Type E sequence is a target of an assay. In some embodiments, the Human adenovirus Type F sequence is a target of an assay. In some embodiments, the Human adenovirus Type G sequence is a target of an assay. In some embodiments, the MERS-CoV sequence is a target of an assay. In some embodiments, the human metapneumovirus sequence is a target of an assay. In some embodiments, the human parainfluenza 1 sequence is a target of an assay. In some embodiments, the human parainfluenza 2 sequence is a target of an assay. In some embodiments, the human parainfluenza 4 sequence is a target of an assay. In some embodiments, the hCoV-OC43 sequence is a target of an assay. In some embodiments, the human parainfluenza 3 sequence is a target of an assay. In some embodiments, the RSV-A sequence is a target of an assay. In some embodiments, the RSV-B sequence is a target of an assay. In some embodiments, the hCoV-229E sequence is a target of an assay. In some embodiments, the hCoV-HKU1 sequence is a target of an assay. In some embodiments, the hCoV-NL63 sequence is a target of an assay. In some embodiments, the Gammacoronavirus sequence is a target of an assay. In some embodiments, the Deltacoronavirus sequence is a target of an assay. In some embodiments, the Alphacoronavirus sequence is a target of an assay. In some embodiments, the Rhinovirus C sequence is a target of an assay. In some embodiments, the Betacoronavirus sequence is a target of an assay. In some embodiments, the Influenza A sequence is a target of an assay. In some embodiments, the Influenza B sequence is a target of an assay. In some embodiments, the SARS-CoV-2 sequence is a target of an assay. In some embodiments, the SARS-CoV-1 sequence is a target of an assay. In some embodiments, the Sarbecovirus subgenus sequence is a target of an assay. In some embodiments, the SARS-related viruses sequence is a target of an assay. In some embodiments, the MS2 sequence is a target of an assay.
In some embodiments, the assay is directed to one or more target sequences. In some embodiments, a target sequence is a portion of an antimicrobial resistance (AMR) gene, such as CTX-M-1, CTX-M-2, CTX-M-25, CTX-M-8, CTX-M-9, or IMP. In some embodiments, a target sequence is a Mycobacterium tuberculosis sequence, such as a portion of IS1081 or IS6110. In some embodiments, a target sequence is an orthopox virus sequence. In some embodiments, a target sequence is a pseudorabies virus sequence. In some embodiments, a target sequence is a Staphylococcus aureus sequence, such as a portion of gyrA or gyrB, or a portion of a S. aureus thermonuclease. In some embodiments, a target sequence is a Stenotrophomonas maltophilia sequence, such as a sequence of S. maltophilia alpha, S. maltophilia beta, or S. maltophilia gamma. In some embodiments, a target sequence is a Bordetalla sp. sequence, such as a sequence of Bordetella bronchoseptica, Bordetella holmesii, Bordetella parapertussis, or Bordetella pertussis. In some embodiments, a target sequence is a Chlamydophila pneumoniae sequence. In some embodiments, a target sequence is a Human adenovirus sequence, such as a sequence of human adenovirus Type A, Type B, Type C, Type D, Type E, Type F, or Type G. In some embodiments, a target sequence is a human bocavirus sequence. In some embodiments, a target sequence is a Legionella pneumophila sequence. In some embodiments, a target sequence is a Mycoplasma pneumoniae sequence. In some embodiments, a target sequence is an Acinetobacter spp. (e.g., A. pitii, A. baumannii, or A. nosocomialis) sequence, such as a portion of gyrB or a 16S-23S ribosomal RNA intergenic spacer sequence. In some embodiments, a target sequence is a Proteus spp. (e.g. P. mirabilis, P. vulgaris, P. penneri, or P. hauseri) sequence, such as a portion of rpoD or 16S. In some embodiments, a target sequence is an Enterobacter spp. (e.g. E. nimipressuralis, E. cloacae, E. asburiae, E. hormaechei, E. kobei, E. ludwigii, or E. mori) sequence, such as a portion of dnaJ, purG, or 16S. In some embodiments, a target sequence is a Bacillus anthracis sequence, such as a portion of pagA or capB. In some embodiments, a target sequence is a Brucella spp. sequence, such as a portion of 23S, bcsp31, or omp2a. In some embodiments, a target sequence is a Coxiella burnetiid sequence, such as a portion of com1 or IS110. In some embodiments, a target sequence is a Francisella tularensis sequence, such as a portion of 16S. In some embodiments, a target sequence is a Rickettsia spp. sequence, such as a portion of 16S, 23S, or 782-17K genus common antigen. In some embodiments, a target sequence is a Yersinia pestis sequence, such as a portion of pMT1, pCD1, or pPCP1. In some embodiments, a target sequence is a A. calcoaceticus sequence, such as a portion of gyrB. In some embodiments, a target sequence is a Francisella tularensis sequence, such as a portion of tul4 or fopA. In some embodiments, a target sequence is a Nocardia spp. sequence, such as a portion of 16S, hsp65, gyrB, secA1, or sodA. In some embodiments, a target sequence is a Cryptococcus spp. sequence, such as a portion of 18S, URA5, ITS, 28S, or CTX1. In some embodiments, a target sequence is an Actinomyces spp. sequence, such as a portion of 16S. In some embodiments, a target sequence is a Streptococcus spp. sequence, such as a portion of 16S, tuf, sodA, or rpoB. In some embodiments, a target sequence is an rRNA sequence, such as a portion of 28S rRNA or 18S rRNA. In some embodiments, a target sequence is a coronavirus sequence, such as a sequence of an alphacoronavirus, betacoronavirus, deltacoronavirus, or gammacoronavirus. In some embodiments, a target sequence is a human coronavirus (hCoV) sequence, such as a sequence of hCoV-229E, hCoV-HKU1, hCoV-NL63, hCoV-OC43. In some embodiments, a target sequence is a MERS-CoV sequence. In some embodiments, the sequence is a mammarenavirus sequence, such as a sequence of a Argentinian mammarenavirus (Junin arenavirus), Lassa mammarenavirus, Lujo mammarenavirus (e.g., an L segment or S segment thereof), or Machupo mammarenavirus. In some embodiments, a target sequence is a human metapneumovirus sequence. In some embodiments, a target sequence is a human parainfluenza sequence, such as a sequence of human parainfluenza 1, human parainfluenza 2, human parainfluenza 3, or human parainfluenza 4. In some embodiments, a target sequence is an influenza A virus sequence, such as a sequence of influenza A H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, N1, N2, N3, N4, N5, N6, N7, N8, or N9. In some embodiments, a target sequence is an influenza B sequence, such as a sequence of influenza B-Victoria V1 or influenza B-Yamagata Y1. In some embodiments, a target sequence is a bacteriophage MS2 sequence. In some embodiments, a target sequence is a rhinovirus C sequence. In some embodiments, a target sequence is a respiratory syncytial virus (RSV) sequence, such as a sequence of RSV-A or RSV-B. In some embodiments, a target sequence is a Sarbecovirus sequence. In some embodiments, a target sequence is a severe acute respiratory syndrome coronavirus (SARS-CoV) sequence, such as a sequence of SARS-CoV-1 or SARS-CoV-2. In some embodiments, a target sequence is a portion of a SARS-CoV-2 S gene, such as a sequence comprising 144/145 wild-type (WT), deletion (del) 144/145 (alpha variant), 156/157 WT, del 156/157 (delta variant), 241/243 WT, del241/243 (beta variant), 69/70 WT, del69/70 (alpha variant), A570 WT, A570D (alpha variant), A701 WT, A701V (beta variant), D1118 WT, D1118H (alpha variant), D215 WT, D215G (beta variant), D614 WT, D614G (beta variant), D80 WT, D80A (beta variant), E484 WT, E484K (gamma variant), P681 WT, P681H (alpha variant), P681R (delta variant), S982 WT, S982A (alpha variant), T19 WT, T19R (delta variant), T716 WT, T716F (gamma variant). In some embodiments, a target sequence is a SARS-related virus sequence. In some embodiments, a target sequence is a portion of a gene selected from 16S, 18S, 23S, 28S, ACTB, ATP5ME, ATP5MF, ATP5MG, ATP5PB, BCSP31, CAPB, CHMP2A, Clorf43, COM1, CTX1, DNAJ, EMC7, FOPA, GPI, GAPDH, GUSB, GYRB, HRPT1, HSP65, NDUFB3, NDUFB4, NDUFB8, OMP2A, PAGA, PRDX1, PSMB2, PSMB4, PURG, RAB7A, REEP5, RNaseP, RPL13, RPL19, RPL27A, RPL30, RPL31, RPL32, RPL37A, RPOB, RPOD, RPS10, RPS27, RPS29, RPS6, SECA1, SNRPD3, SODA, TUF, TUL4, URA5, VCP, VPS29, and YWHAG.
In some embodiments, the one or more targets may be at a concentration of 1 copy/reaction, at least about 2 copies/reaction, at least about 3 copies/reaction, at least about 4 copies/reaction, at least about 5 copies/reaction, at least about 6 copies/reaction, at least about 7 copies/reaction, at least about 8 copies/reaction, at least about 9 copies/reaction, at least about 10 copies/reaction, at least about 20 copies/reaction, at least about 30 copies/reaction, at least about 40 copies/reaction, at least about 50 copies/reaction, at least about 60 copies/reaction, at least about 70 copies/reaction, at least about 80 copies/reaction, at least about 90 copies/reaction, at least about 100 copies/reaction, at least about 200 copies/reaction, at least about 300 copies/reaction, at least about 400 copies/reaction, at least about 500 copies/reaction, at least about 600 copies/reaction, at least about 700 copies/reaction, at least about 800 copies/reaction, at least about 900 copies/reaction, at least about 1000 copies/reaction, at least about 2000 copies/reaction, at least about 3000 copies/reaction, at least about 4000 copies/reaction, at least about 5000 copies/reaction, at least about 6000 copies/reaction, at least about 7000 copies/reaction, at least about 8000 copies/reaction, at least about 9000 copies/reaction, at least about 10000 copies/reaction, at least about 20000 copies/reaction, at least about 30000 copies/reaction, at least about 40000 copies/reaction, at least about 50000 copies/reaction, at least about 60000 copies/reaction, at least about 70000 copies/reaction, at least about 80000 copies/reaction, at least about 90000 copies/reaction, or at least about 100000 copies/reaction.
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, CDKN1 C, 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, RAD51 C, 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.
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, DCLRE1 C, 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, POMGNTI, PPT1, PROP1, PRPS1, PSAP, PTS, PUS1, PYGM, RAB23, RAG2, RAPSN, RARS2, RDH12, RMRP, RPE65, RPGRIPIL, 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, USH1 C, USH2A, VPS13A, VPS13B, VPS45, VRK1, VSX2, WNT10A, XPA, XPC, and ZFYVE26.
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.
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.
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.
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).
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
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.
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 101 non-target nucleic acids, 102 non-target nucleic acids, 103 non-target nucleic acids, 104 non-target nucleic acids, 105 non-target nucleic acids, 106 non-target nucleic acids, 107 non-target nucleic acids, 108 non-target nucleic acids, 109 non-target nucleic acids, or 1010 non-target nucleic acids.
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 101 non-target nucleic acids, 102 non-target nucleic acids, 103 non-target nucleic acids, 104 non-target nucleic acids, 105 non-target nucleic acids, 106 non-target nucleic acids, 107 non-target nucleic acids, 108 non-target nucleic acids, 109 non-target nucleic acids, or 1010 non-target nucleic acids. The target nucleic acid populations can be present at different concentrations or amounts in the sample.
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.
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 and to enhance the detection capabilities of the devices of the present disclosure. In some cases, the sample is not divided into subsamples.
In some embodiments, the sample can be provided manually to the detection device of the present disclosure. For example, a swab sample can be dipped into a solution and the sample/solution can be pipetted into the device. In other embodiments, the sample can be provided via an automated syringe. The automated syringe can be configured to control a flow rate at which the sample is provided to the detection device. The automated syringe can be configured to control a volume of the sample that is provided to the detection device over a predetermined period.
In some embodiments, the sample can be provided directly to the detection device of the present disclosure. For example, a swab sample can be inserted into a sample chamber on the detection device.
The sample can be prepared before one or more targets are detected within the sample. The sample preparation steps described herein can process a crude sample to generate a pure or purer sample. Sample preparation can one or more physical or chemical processes, including, for example, nucleic acid purification, lysis, binding, washing, and/or eluting. In certain instances, sample preparation can comprise the following steps, in any order, including sample collection, nucleic acid purification, heat inactivation, enzyme inactivation, and/or base/acid lysis.
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).
In some embodiments, lysis of a collected sample can be performed. Lysis can be performed using a protease (e.g., a Proteinase K or PK enzyme). Exemplary proteases include serine proteases (e.g., Proteinase K, Savinase®, trypsin, Protamex®, etc.), metalloproteinases (e.g., MMP-3, etc.), cysteine proteases (e.g, cathepsin B, papin, etc.), threonine proteases, aspartic proteases (e.g., renin, pepsin, cathepsin D, etc.), glutamic proteases, asparagine peptide lyases, or the like. In some cases, a solution of reagents can be used to lyse the cells in the sample and release the nucleic acids so that they are accessible to the programmable nuclease. Active ingredients of the solution can be chaotropic agents, detergents, salts, and can be of high osmolality, ionic strength, and pH. Chaotropic agents or chaotropes are substances that disrupt the three-dimensional structure in macromolecules such as proteins, DNA, or RNA. One example protocol may comprise a 4 M guanidinium isothiocyanate, 25 mM sodium citrate·2H20, 0.5% (w/v) sodium lauryl sarcosinate, and 0.1 M β-mercaptoethanol), but numerous commercial buffers for different cellular targets can also be used. Alkaline buffers can also be used for cells with hard shells, particularly for environmental samples. Detergents such as sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB) can also be implemented to chemical lysis buffers. Cell lysis can also be performed by physical, mechanical, thermal or enzymatic means, in addition to chemically-induced cell lysis mentioned previously. In some cases, depending on the type of sample, nanoscale barbs, nanowires, acoustic generators, integrated lasers, integrated heaters, and/or microcapillary probes can be used to perform lysis.
In certain instances, heat inactivation can be performed on the sample. In some embodiments, a processed/lysed sample can undergo heat inactivation to inactivate, in the lysed sample, the proteins used during lysing (e.g, a PK enzyme or a lysing reagent) and/or other residual proteins in the sample (e.g., RNases, DNases, viral proteins, etc.). In some cases, a heating element integrated into 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.
In certain instances, enzyme inactivation can be performed on the sample. In some embodiments, a processed/lysed sample can undergo enzyme inactivation to inhibit or inactivate, in the lysed sample, the proteins used during lysing (e.g., a PK enzyme or a lysing reagent) and/or other residual proteins in the sample (e.g., RNases, DNases, etc.). In some cases, a solution of reagents can be used to inactivate one or more enzymes present in the sample. Enzyme inactivation can occur before, during, or after lysis, when lysis is performed. For example, an RNase inhibitor may be included as a lysis reagent to inhibit native RNases within the sample (which might otherwise impair target and/or reporter detection downstream). Exemplary RNase inhibitors include RNAse Inhibitor, Murine (NEB), RnaseIn Plus (Promega), Protector Rnase Inhibitor (Roche), SuperaseIn (Ambion), RiboLock (Thermo), Ribosafe (Bioline), or the like. Alternatively, or in combination, when a protease is used for sample lysis, a protease inhibitor can be applied to the lysed sample to inactivate the protease prior to contacting the sample nucleic acids to the programmable nuclease. Additional application of heat may not be required to inhibit the protease (e.g., proteinase K) sufficiently to prevent additional activity of the protease (which could potentially impair programmable nuclease activity downstream, in some embodiments). Exemplary protease inhibitors include AEBSF, antipain, aprotinin, bestatin, chymostatin, EDTA, leupeptin, pepstatin A, phosphoramidon, PMSF, soybean trypsin inhibitor, TPCK, or the like. In some instances, enzyme inactivation may occur before, during, after, or instead of heat inactivation.
In some cases, a target nucleic acid within the sample can undergo amplification before binding to a guide nucleic acid, for example a crRNA of a CRISPR enzyme. The target nucleic acid within a purified sample can be amplified. In some instances, amplification can be accomplished using loop mediated amplification (LAMP), isothermal recombinase polymerase amplification (RPA), and/or polymerase chain reaction (PCR). 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 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 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.
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. 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.
Additionally, target nucleic acid can optionally be amplified before binding to the guide nucleic acid (e.g., crRNA) 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 RNA. 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.
In some cases, the movable mechanism can comprise a plurality of valves. The plurality of valves can comprise, for example, a check valve. In some cases, the movable mechanism can comprise a plunger or a bristle. The plunger or bristle can have an open configuration and a closed configuration. As described above, the open configuration can permit a continuous flow of the sample through one or more sections of the channel, and the closed configuration can restrict or completely inhibit a flow of the sample through one or more sections of the channel. The closed configuration can permit a physical and/or thermal separation of one or more volumes or portions of the sample flowing through the channel. When in the open configuration, the plunger or the bristle can be positioned flush to a bottom of the channel so that the sample can flow through the channel. When in the closed configuration, the plunger or the bristle can be configured to extend from a bottom portion of the channel to a top portion of the channel so that the sample flow is restricted and the sample is divided into a plurality of different droplets, or partitions, that are physically and/or thermally isolated from each other. In some cases, the movable mechanism can comprise any physical object (e.g., a plate) that can be configured to restrict flow through the channel at one or more sections of the channel. In some cases, the movable mechanism can comprise a hinge or spring mechanism to move the movable mechanism between an open configuration and a closed configuration.
The movable mechanisms can be used to generate one or more droplets, aliquots, or subsamples. Each of the one or more droplets, aliquots, or subsamples generated using the movable mechanism can be physically and/or thermally isolated within a plurality of different portions within the channel. The droplets, aliquots, or subsamples can be physically constrained within different portions within the channel. The droplets, aliquots, or subsamples can be constrained between a first movable mechanism that is in a closed position and a second movable mechanism that is in a closed position. The first movable mechanism can be located at a first distance from an inlet of the channel, and the second movable mechanism can be located at a second distance from the inlet of the channel. The channel can be part of a closed system through which the sample can flow. In some cases, when the sample flow through an inlet of the channel is stopped (e.g., a plunger of a syringe containing the sample is pulled back), the one or more movable mechanisms can be placed in a closed configuration, thereby separating the sample already within the channel into a plurality of thermally and physically isolated droplets. Generating droplets, aliquots, or subsamples can simplify the solution, reduce a complexity of the solution, and enhance an accessibility of targets for amplification.
The one or more droplets, aliquots, or subsamples generated using the movable mechanism can undergo an amplification step or a thermocycling step as described elsewhere herein. In some cases, the one or more droplets generated using the movable mechanisms can come into contact with separate heating units and heat sinks while constrained between two movable mechanisms. Different sections of the channel can comprise a plurality of heating units and heat sinks configured to perform thermocycling for different droplets. Individual thermocycling of the droplets, aliquots, or subsamples can permit more efficient thermocycling of smaller volumes of fluid, and can require less energy usage (e.g., from a battery). One or more valves can control a flow or a movement of the sample through the channel. The one or more valves can comprise a check valve that is configured to restrict a movement of the sample or the one or more droplets such that the sample or the one or more droplets do not travel backwards towards an inlet portion of the channel. The one or more valves can control when the sample or the droplets come into thermal contact with the heating unit and/or the heat sink. The timing of such thermal contact can correspond to a timing of one or more thermocycling steps. In some cases, a first droplet of the sample can be in thermal contact with a first heating unit and a first heat sink, a second droplet of the sample can be in thermal contact with a second heating unit and a second heat sink, and so on.
As described above, the 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 the one or more movable mechanisms described above. In some cases, each droplet of the sample can undergo one or more sample preparation steps (e.g., nucleic acid purification, lysis, heat inactivation, enzyme inactivation, amplification, etc.) independently and/or in parallel while the droplets are physically constrained or thermally isolated between two movable mechanisms.
After amplification, the sample can be remixed. The sample can be circulated through the detection chamber using a bulk circulation mechanism that is configured to stir the remixed sample around such that the remixed sample comes into contact with one or more programmable nuclease probes, as shown in
In some embodiments, electrowetting can be used by the device for sample transport. In some cases, the device can be configured for electrowetting-on-dielectric (EWOD) applications. The devices of the present disclosure can comprise an array of independently addressable electrodes integrated into the device.
Described herein are various embodiments of a device for programmable nuclease-based (e.g., CRISPR-based) assays.
In certain instances, as seen in
The programmable nuclease probe can comprise a programmable nuclease and/or a guide nucleic acid. The guide nucleic acid can bind to a target nucleic acid, as described in greater detail below. In some case, to minimize off-target binding (which can slow down detection or inhibit accurate detection), the device can be configured to generate an electro-potential gradient or to provide heat energy to one or more regions proximal to the programmable nuclease probe, to enhance targeting.
In some embodiments, one or more guide nucleic acids can be used to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein. The guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein. The guide nucleic acid is complementary to the target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid can be a RNA, DNA, or synthetic nucleic acids. A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. A guide nucleic acid can be a crRNA. Sometimes, a guide nucleic acid may comprise a crRNA and tracrRNA. The guide nucleic acid can bind specifically to the target nucleic acid. In some cases, the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids. The target nucleic acid can be designed and made to provide desired functions. In some cases, the targeting region of a guide nucleic acid is 20 nucleotides in length. The targeting region of the guide nucleic acid can have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some instances, the targeting region of the guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or 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.
The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest. The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV 18. Often, guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein. The tiling, for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling may comprise gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential. Often, a method for detecting a target nucleic acid may comprise contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some detector nucleic acids of a population of detector nucleic acids. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that can be caused by multiple organisms.
In some embodiments, programmable nucleases can be used to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. A programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. Often, the signal is present prior to detector nucleic acid cleavage and changes upon detector nucleic acid cleavage. Sometimes, the signal is absent prior to detector nucleic acid cleavage and is present upon detector nucleic acid cleavage. The 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).
The programmable nuclease system used to detect modified target nucleic acids can comprise CRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), Cas proteins, and detector nucleic acids.
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.
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 V1 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 V1 CRISPR/Cas enzyme.
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 Cas 12 nuclease can be a Cas12a (also referred to as Cpf1) protein, a Cas12b protein, Cas 12c protein, Cas12d protein, or a Cas12e protein.
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 having the sequence of SEQ ID NO: 17 (or a sequence with at least 60%, 70%, 80%, 90%, or 95% identity thereto). In some cases, the programmable nuclease 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 V1 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.
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 V1 CRISPR/Cas enzyme (e.g., a Cas13 nuclease). For example, Cas 13a 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 V1 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.
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 V1 protein, with a DNA reporter and an RNA reporter, can enable multiplexed detection of target ssDNAs or a combination of a target dsDNA and a target ssDNA, respectively. Multiplexing of different RNA-activated programmable RNA nucleases that have distinct RNA reporter cleavage preferences can enable additional multiplexing. Methods for the generation of ssDNA for DNA-activated programmable RNA nuclease-based diagnostics can include (1) asymmetric PCR, (2) asymmetric isothermal amplification, such as RPA, LAMP, SDA, etc. (3) NEAR for the production of short ssDNA molecules, and (4) conversion of RNA targets into ssDNA by a reverse transcriptase followed by RNase H digestion. Thus, DNA-activated programmable RNA nuclease detection of target DNA is compatible with the various systems, kits, compositions, reagents, and methods disclosed herein. For example, target ssDNA detection by Cas13a can be employed in a detection device as disclosed herein.
In any of the embodiments described herein, the programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid, which can initiate trans cleavage activity. In some cases, the trans cut or trans cleavage can cut and/or release a reporter. In other cases, the trans cut or trans cleavage can produce an analog of a target, which can be directly detected. Trans cleavage activity can be non-specific cleavage of nearby nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal. For example, the detection moiety can correspond to the element, or moiety, (X) shown in
Reporters, which can be referred to interchangeably as reporters or detector nucleic acids, can be used in conjunction with the compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, etc.) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. The reporter can be suspended in solution or immobilized on a surface. For example, the reporter can be immobilized on the surface of a chamber in a device as disclosed herein. In some cases, the reporter can be immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they are held in position by a magnet placed below the chamber. The reporter can be capable of being cleaved by the activated programmable nuclease, thereby generating a detectable signal. The detectable signal can correspond to a release of one or more elements (X) as illustrated in
As used herein, a detector nucleic acid is used interchangeably with reporter or reporter. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the detector nucleic acid is a single-stranded nucleic acid comprising ribonucleotides. The detector nucleic acid can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the detector nucleic acid may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some cases, the detector nucleic acid has only ribonucleotide residues. In some cases, the detector nucleic acid has only deoxyribonucleotide residues. In some cases, the detector nucleic acid may comprise nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the detector nucleic acid may comprise synthetic nucleotides. In some cases, the detector nucleic acid may comprise at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the detector nucleic acid is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length. In some cases, the detector nucleic acid is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length. In some cases, the detector nucleic acid may comprise at least one uracil ribonucleotide. In some cases, the detector nucleic acid may comprise at least two uracil ribonucleotides. Sometimes the detector nucleic acid has only uracil ribonucleotides. In some cases, the detector nucleic acid may comprise at least one adenine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two adenine ribonucleotides. In some cases, the detector nucleic acid has only adenine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one cytosine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two cytosine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one guanine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two guanine ribonucleotides. A detector nucleic acid can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the detector nucleic acid is from 5 to 12 nucleotides in length. In some cases, the detector nucleic acid is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the detector nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. For cleavage by a programmable nuclease comprising Cas13, a detector nucleic acid can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Cas12, a detector nucleic acid can be 10 nucleotides in length.
The single stranded detector nucleic acid can comprise a detection moiety capable of generating a first detectable signal. Sometimes the detector nucleic acid may comprise a protein capable of generating a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorometric, etc.), or piezo-electric signal. In some cases, a detection moiety is on one side of the cleavage site. Optionally, a quenching moiety is on the other side of the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety. In some cases, the quenching moiety is 5′ to the cleavage site and the detection moiety is 3′ to the cleavage site. In some cases, the detection moiety is 5′ to the cleavage site and the quenching moiety is 3′ to the cleavage site. Sometimes the quenching moiety is at the 5′ terminus of the detector nucleic acid. Sometimes the detection moiety is at the 3′ terminus of the detector nucleic acid. In some cases, the detection moiety is at the 5′ terminus of the detector nucleic acid. In some cases, the quenching moiety is at the 3′ terminus of the detector nucleic acid. In some cases, the single-stranded detector nucleic acid is at least one population of the single-stranded nucleic acid capable of generating a first detectable signal. In some cases, the single-stranded detector nucleic acid is a population of the single stranded nucleic acid capable of generating a first detectable signal. Optionally, there are more than one population of single-stranded detector nucleic acid. In some cases, there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, or greater than 50, or any number spanned by the range of this list of different populations of single-stranded detector nucleic acids capable of generating a detectable signal. In some cases, there are from 2 to 50, from 3 to 40, from 4 to 30, from 5 to 20, or from 6 to 10 different populations of single-stranded detector nucleic acids capable of generating a detectable signal. In some cases there are from 2 to 50, from 5 to 50, from 10 to 50, from 15 to 50, from 20 to 50, from 25 to 50, from 30 to 50, from 35 to 50, from 40 to 50, from 2 to 40, from 5 to 40, from 10 to 40, from 15 to 40, from 20 to 40, from 25 to 40, from 30 to 40, from 35 to 40, from 2 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, from 2 to 20, from 5 to 20, from 10 to 20, from 15 to 20, from 2 to 10, or from 5 to 10 different populations of single-stranded detector nucleic acids capable of generating a detectable signal.
In some embodiments, target nucleic acid amplicons are detected by immobilized programmable nuclease probes, such as, for example, CRISPR CAS guide RNA probes (referred to as CRISPR probe). Upon a complementary binding event between a target nucleic acid amplicon and a programmable nuclease probe (e.g., an immobilized CRISPR CAS/guide RNA complex) a cutting event will occur that release a reporter that is then detected by a sensor. There are two main schemes for detection of the binding events between the target and the programmable nuclease probe, a mobile phase scheme as illustrated in
In certain circumstances, a mobile phase detection scheme (
In some embodiments, a stationary phase detection scheme is used. In these embodiments the remixed sample can contain one or more target nucleic acid amplicon sequence types and copies thereof.
The plurality of programmable nuclease probes shown in
As described above, the single stranded detector nucleic acid can comprise a detection moiety capable of generating a first detectable signal. Sometimes the detector nucleic acid may comprise a protein capable of generating a signal. A signal can be a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorometric, 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, colorometric, etc.), or piezo-electric signal. A detector nucleic acid, sometimes, is protein-nucleic acid that can generate a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorometric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. Often a calorimetric signal is heat produced after cleavage of the detector nucleic acids. Sometimes, a calorimetric signal is heat absorbed after cleavage of the detector nucleic acids. A potentiometric signal, for example, is electrical potential produced after cleavage of the detector nucleic acids. An amperometric signal can be movement of electrons produced after the cleavage of detector nucleic acid. Often, the signal is an optical signal, such as a colorometric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the detector nucleic acids. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of detector nucleic acids. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the detector nucleic acid.
Detecting the presence or absence of a target nucleic acid of interest can involve measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The signal can be measured using one or more sensors integrated with the device or operatively coupled to the device. Thus, the detecting steps disclosed herein can involve measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, a signal can be present in the absence of detector nucleic acid cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal can be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
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, colorometric, 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 detector nucleic acid. 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 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 a digital camera, such a digital camera on a mobile device. The mobile device can have a software program or a mobile application that can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. Any suitable detection or measurement 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).
In certain aspects of this disclosure, multiplexing refers to parallel sensing of multiple target nucleic acid sequences in one sample by multiple probes.
The devices of the present disclosure can be used to implement for detection of one or more target nucleic acids within the sample. The devices of the present disclosure can comprise one or multiple pumps, valves, reservoirs, and chambers for sample preparation, optional amplification of a target nucleic acid within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by a programmable nuclease.
Methods consistent with the present disclosure include a multiplexing method of assaying for a plurality of target nucleic acids in a sample. A multiplexing method may comprise contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some reporters (e.g., protein-nucleic acids) of a population of reporter molecules (e.g., protein-nucleic acids), wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample.
Multiplexing can comprise spatial multiplexing wherein multiple different target nucleic acids are detected at the same time, but the reactions are spatially separated. In some cases, the multiple target nucleic acids are detected using the same programmable nuclease, but different guide nucleic acids. The multiple target nucleic acids sometimes are detected using the different programmable nucleases. In the case wherein multiple target nucleic acids are detected using the different programmable nucleases, the method involves using a first programmable nuclease, which upon activation (e.g., after binding of a first guide nucleic acid to a first target), cleaves a nucleic acid of a first reporter and using a second programmable nuclease, which upon activation (e.g., after binding of a second guide nucleic acid to a second target), cleaves a nucleic acid of a second reporter.
Sometimes, multiplexing can be single reaction multiplexing wherein multiple different target acids are detected in a single reaction volume. Often, at least two different programmable nucleases are used in single reaction multiplexing. For example, multiplexing can be enabled by immobilization of multiple categories of detector nucleic acids within a fluidic system, to enable detection of multiple target nucleic acids within a single fluidic system. Multiplexing allows for detection of multiple target nucleic acids in one kit or system. In some cases, the multiple target nucleic acids comprise different target nucleic acids to a virus, a bacterium, or a pathogen responsible for one disease. In some cases, the multiple target nucleic acids comprise different target nucleic acids associated with a cancer or genetic disorder. Multiplexing for one disease, cancer, or genetic disorder increases at least one of sensitivity, specificity, or accuracy of the assay to detect the presence of the disease in the sample. In some cases, the multiple target nucleic acids comprise target nucleic acids directed to different viruses, bacteria, or pathogens responsible for more than one disease. In some cases, multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes of the same bacteria or pathogen responsible for a disease, for example, for a wild-type genotype of a bacteria or pathogen and for genotype of a bacteria or pathogen comprising a mutation, such as a single nucleotide polymorphism (SNP) that can confer resistance to a treatment, such as antibiotic treatment. Multiplexing, thus, allows for multiplexed detection of multiple genomic alleles. For example, multiplexing may comprise method of assaying comprising a single assay for a microorganism species using a first programmable nuclease and an antibiotic resistance pattern in a microorganism using a second programmable nuclease. Sometimes, multiplexing allows for discrimination between multiple target nucleic acids of different HPV strains, for example, HPV16 and HPV18. In some cases, the multiple target nucleic acids comprise target nucleic acids directed to different cancers or genetic disorders. Often, multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes, for example, for a wild-type genotype and for SNP genotype. Multiplexing for multiple diseases, cancers, or genetic disorders provides the capability to test a panel of diseases from a single sample. For example, multiplexing for multiple diseases can be valuable in a broad panel testing of a new patient or in epidemiological surveys. Often multiplexing is used for identifying bacterial pathogens in sepsis or other diseases associated with multiple pathogens.
Furthermore, signals from multiplexing can be quantified. For example, a method of quantification for a disease panel may comprise assaying for a plurality of unique target nucleic acids in a plurality of aliquots from a sample, assaying for a control nucleic acid control in a second aliquot of the sample, and quantifying a plurality of signals of the plurality of unique target nucleic acids by measuring signals produced by cleavage of detector nucleic acids compared to the signal produced in the second aliquot. In this context, a unique target nucleic acid refers to the sequence of a nucleic acid that has an at least one nucleotide difference from the sequences of the other nucleic acids in the plurality. Multiple copies of each target nucleic acid can be present. For example, a unique target nucleic population can comprise multiple copies of the unique target nucleic acid. Often the plurality of unique target nucleic acids is from a plurality of bacterial pathogens in the sample.
In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 2 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 3 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 4 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 5 different target nucleic acids in a single reaction. In some cases, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single reaction. In some instances, the multiplexed kits detect at least 2 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 3 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 4 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 5 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect 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, from 9 to 10, from 2 to 9, from 3 to 9, from 4 to 9, from 5 to 9, from 6 to 9, from 7 to 9, from 8 to 9, from 2 to 8, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 7, from 3 to 7, from 4 to 7, from 5 to 7, from 6 to 7, from 2 to 6, from 3 to 6, from 4 to 6, from 5 to 6, from 2 to 5, from 3 to 5, from 4 to 5, from 2 to 4, from 3 to 4, or from 2 to 3 different target nucleic acids in a single kit. Multiplexing can be carried out in a single-pot or “one-pot” reaction, where reverse transcription, amplification, in vitro transcription, or any combination thereof, and detection are carried out in a single volume. Multiplexing can be carried out in a “two-pot reaction”, where reverse transcription, amplification, in vitro transcription, or any combination thereof, are carried out in a first volume and detection is carried out in a second volume.
In some cases, multiplexing can comprise detecting multiple targets with a single probe. Alternatively, multiplexing can comprise detecting multiple targets with multiple probes. The multiple probes can be configured to detect a presence of a particular sequence, target nucleic acid, or a plurality of different target sequences or nucleic acids.
The devices of the present disclosure can be manufactured from a variety of different materials. Exemplary materials that can be used include plastic polymers, such as poly-methacrylate (PMMA), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene (PE), high-density polyethylene (HDPE), polypropylene (PP); glass; and silicon. Features of the device (e.g., chambers, channels, etc.) can be manufactured by various processes. For example, the features can be (1) embossed using injection molding, (2) micro-milled or micro-engraved using computer numerical control (CNC) micromachining or non-contact laser drilling (by means of a CO2 laser source); (3) generated using additive manufacturing, and/or (4) generated using one or more photolithographic or stereolithographic methods.
In some embodiments, any of the devices of the present disclosure can comprise a sample interface configured to receive a sample that may comprise at least one gene of interest. The device can further comprise a channel in fluid communication with the sample interface and a detection chamber. In some cases, the channel may comprise one or more movable mechanisms to separate the sample into a plurality of droplets. 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 probe disposed on a surface of said detection chamber. The at least one programmable nuclease probe can comprise a guide nucleic acid complexed with a programmable nuclease. In some cases, the programmable nuclease probe may comprise a CRISPR/Cas enzyme. In some cases, the guide nucleic acid may comprise a guide RNA. In some embodiments, the device may comprise a plurality of programmable nuclease probes comprising different guide RNAs.
The device can further comprise a plurality of sensors that determine a presence of said at least one gene of interest by detecting a signal produced upon cleavage of a target nucleic acid region in said at least one gene of interest by said at least one programmable nuclease probe. The cleavage of the target nucleic acid region can occur after a complementary binding of said target nucleic acid region to said guide nucleic acid of said at least one programmable nuclease probe.
As described elsewhere herein, the one or more movable mechanisms can comprise one or more valves configured to restrict flow through one or more sections of the channel. The one or more movable mechanisms can comprise a plunger or a bristle that is configured to restrict flow through one or more sections of the channel. The one or more movable mechanisms can be operatively coupled to a power source that is integrated with or insertable into the device. The power source can comprise a battery.
In some cases, any of the devices described herein may comprise a physical filter to filter one or more particles from the sample that do not comprise the one or more targets (e.g., a gene of interest). In some cases, the device may comprise a sample preparation chamber. The sample preparation chamber can comprise a lysing agent. The sample preparation chamber can comprise a heating unit configured for heat inactivation. The sample preparation chamber can comprise one or more reagents for nucleic acid purification.
In some cases, the channel between the sample interface and the detection chamber may comprise a plurality of heating elements and a plurality of heat sinks for amplifying the at least one gene of interest or a portion thereof. The plurality of heating elements and the plurality of heat sinks can be configured to perform one or more thermocycling operations on the sample or at least a portion of the sample (e.g., the plurality of droplets).
As described elsewhere herein, the signal produced upon cleavage of a target nucleic acid can be associated with a physical, chemical, or electrochemical change or reaction. The signal can comprise an optical signal, a fluorescent or colorimetric signal, a potentiometric or amperometric signal, and/or a piezo-electric signal. In some cases, the signal is associated with a change in an index of refraction of a solid or gel volume in which the at least one programmable nuclease probe is disposed.
In some embodiments, the device may comprise a sample interface configured to receive a sample that may comprise one or more genomic targets of interest. In some cases, the one or more genomic targets of interest comprise a sequence of nucleic acids comprising the nucleic acid.
The device can further comprise one or more channels comprising one or more movable mechanisms to separate the sample into partitioned samples. The one or more channels can be in fluid communication with the sample interface and a reaction chamber that is configured to receive and contact the partitioned samples with an enzyme, reagent, or programmable detection agent that is configured to cleave a nucleic acid of said one or more genomic targets of interest.
The device can further comprise a plurality of sensors for determining a presence of the one or more genomic targets of interest by detecting one or more reporters released by said cleavage of said nucleic acid. The programmable detection agent can be a CRISPR/Cas enzyme. In some cases, the reporter may comprise a nucleic acid and a detection moiety. In some cases, the reporter may comprise at least one ribonucleotide or at least one deoxyribonucleotide. In some cases, the reporter may comprise a DNA nucleic acid or an RNA nucleic acid. The reported molecule can be immobilized on a surface of the detection chamber (i.e., a movement of the reporter can be physically or chemically constrained).
In some cases, the one or more movable mechanisms comprise a plurality of valves configured to restrict flow in a first direction through the one or more channels towards the sample interface. The plurality of valves can be configured to selectively permit flow in a second direction through the one or more channels towards the reaction chamber. A first valve and a second valve of the plurality of valves can be configured to physically, fluidically, or thermally isolate a first portion of the sample from a second portion of the sample when the first valve and the second valve are in a closed state.
The one or more channels can comprise a plurality of heating elements and a plurality of heat sinks to perform thermocycling on the partitioned samples. A first heating element of the plurality of heating elements and a first heat sink of the plurality of heat sinks can be positioned between a first movable mechanism and a second movable mechanism of the one or more movable mechanisms.
In any of the embodiments described herein, the device can further comprise a telemedicine unit configured to provide one or more detection results to a computing unit that is remote from the device. In some embodiments, the telemedicine unit provides one or more detection results to a computing unit that is remote to the device through a cloud-based connection. In some embodiments, the telemedicine unit is HIPAA compliant. In some embodiments, the telemedicine unit transmits encrypted data. The computing unit can comprise a mobile device or a computer. The one or more detection results can indicate a presence or an absence of a target nucleic acid of interest in the sample.
In another aspect, the present disclosure provides a method for target detection. The method can comprise contacting a sample with the device of any of the preceding claims and detecting a presence or an absence of one or more genes of interest in said sample. In some cases, the method can comprise generating one or more detection results indicating the presence or the absence of the one or more genes of interest in the sample. In some cases, the method can comprise transmitting the one or more detection results to a remote computing unit. The remote computing unit can comprise, for example, a mobile device.
In another aspect, the present disclosure provides a method for target detection. The method can comprise providing a sample comprising at least one gene of interest. The method can comprise separating the sample into a plurality of sub-samples using one or more movable mechanisms described herein. The method can comprise receiving the plurality of sub-samples in a detection chamber and contacting the plurality of sub-samples with at least one programmable nuclease probe disposed on a surface of said detection chamber. The at least one programmable nuclease probe can comprise a guide nucleic acid complexed with a programmable nuclease. In some cases, the method can comprise contacting the plurality of sub-samples with a plurality of programmable nuclease probes comprising different guide RNAs. The method can comprise using a plurality of sensors to determine a presence or an absence of said at least one gene of interest by detecting a signal produced upon cleavage of a target nucleic acid region in said at least one gene of interest by said at least one programmable nuclease probe.
In some cases, the method can further comprise amplifying the at least one gene of interest after separating the sample into a plurality of sub-samples. In some cases, the method can further comprise amplifying the at least one gene of interest before mixing the plurality of sub-samples in the detection chamber. Amplifying the at least one gene of interest can comprise using a plurality of heating elements and a plurality of heat sinks to perform thermocycling on the plurality of sub-samples.
In some cases, the method can comprise using a physical filter to filter one or more particles from the sample that do not comprise the one or more target genes of interest. In some cases, the method can comprise lysing the sample before detecting the one or more target genes of interest. In some cases, the method can comprise performing heat inactivation on the sample. In some cases, the method can comprise performing enzyme (e.g., PK) inactivation on the sample. In some cases, the method can comprise performing nucleic acid purification on the sample.
In some cases, the detection devices described herein can be configured to implement process control procedures to ensure that the sample preparation, target amplification, and target detection processes are performed accurately and as intended.
Described herein are various embodiments, for point of need (PON) programmable nuclease-based devices. In some embodiments, the PON device is configured for a 5-plex respiratory panel as shown in
Described herein are various Point-of-need (PON) diagnostics that can rapidly identify causes of ailments on location where needed by patients. In some embodiments, disposable PON devices can be manufactured and delivered to the user in a sterile condition to help fill this need. However, in some embodiments, transferring assays from the lab to a point-of-need device can be a challenge. In some embodiments, integrating a fluidic system capable of delivering rapid, reliable, and easy to interpret results while still being disposable is especially challenging.
Disclosed herein are various embodiments of methods, devices, and compositions for a disposable fluidic workflow. In some embodiments, the workflow method may comprise: (1) sample collection from the patient and delivery to the device, (2) lysis, (3) amplification, and (4) detection/readout. In some embodiments, any of the disposable fluidic devices described herein, may comprise an exterior housing and an interior control printed circuit board (PCB) on which to mount sub-components. In some embodiments, such subcomponents may comprise a swab to collect a sample from patient, a mechanism for extracting sample from the swab (e.g., a scraper), fluidics configured to move the sample within the device, one or more sample chambers, one or more reagent storage bags or chambers, amplification mechanisms, detectors, valves and heating elements. In some embodiments, described herein, valves may be rotary valves capable of diverting sample to and from one chamber or channel to multiple other chambers or channels.
The present disclosure provides various devices, systems, fluidic devices, and kits for rapid tests, which may quickly assess whether a target nucleic acid is present in a sample by using a programmable nuclease that can interact with functionalized surfaces of the fluidic systems to generate a detectable signal. For example, disclosed herein are particular microfluidic devices, lateral flow devices, sample preparation devices, and compositions (e.g., programmable nucleases, guide RNAs, reagents for in vitro transcription, amplification, reverse transcription, and reporters, or any combination thereof) for use in said devices that are particularly well suited to carry out a highly efficient, rapid, and accurate reactions for detecting the presence of a target nucleic acid (e.g., a DETECTR reaction). The systems and programmable nucleases disclosed herein can be used as a companion diagnostic with any of the diseases disclosed herein (e.g., RSV, sepsis, flu, COVID-19), or can be used in reagent kits, point-of-care diagnostics, or over-the-counter diagnostics. The systems may be used as a point of care diagnostic or as a lab test for detection of a target nucleic acid and, thereby, detection of a condition, for example, in a subject from which the sample was taken. The systems may be used in various sites or locations, such as in laboratories, in hospitals, in physician offices/laboratories (POLs), in clinics, at remotes sites, or at home. Sometimes, the present disclosure provides various devices, systems, fluidic devices, and kits for consumer genetic use or for over-the-counter use.
Guide nucleic acids are compatible for use in the devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, and lateral flow devices) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, and reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample (e.g., DETECTR reactions). The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein. The guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein. The guide nucleic acid is complementary to the target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid may be a RNA, DNA, or synthetic nucleic acids. A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. A guide nucleic acid can be a crRNA. Sometimes, a guide nucleic acid may comprise a crRNA and tracrRNA. The guide nucleic acid can bind specifically to the target nucleic acid. In some cases, the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids. The target nucleic acid can be designed and made to provide desired functions. In some cases, the targeting region of a guide nucleic acid is 20 nucleotides in length. The targeting region of the guide nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some instances, the targeting region of the guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable or bind specifically. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid. The 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.
The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest. The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV 18. Often, guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein. The tiling, for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling may comprise gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential. Often, a method for detecting a target nucleic acid may comprise contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some detector nucleic acids of a population of detector nucleic acids. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that may be caused by multiple organisms. Exemplary guide nucleic acids are listed in Table 2. In Table 2, references to a “Cas12 Variant” refer to a Cas12 variant having the sequence of SEQ ID NO: 17, and references to “CasM.26” refer to a Cas13 variant having the sequence of SEQ ID NO: 21.
Sarbecovirus subgenus
Sarbecovirus subgenus
Sarbecovirus subgenus
Bordetella parapertussis
Bordetella parapertussis
Bordetella parapertussis
Bordetella pertussis
Bordetella pertussis
Bordetella pertussis
Bordetella bronchoseptica
Bordetella bronchoseptica
Bordetella bronchoseptica
Bordetella holmesii
Bordetella holmesii
Bordetella holmesii
Chlamydophila pneumoniae
Chlamydophila pneumoniae
Chlamydophila pneumoniae
Legionella pneumophila
Legionella pneumophila
Legionella pneumophila
Mycoplasma pneumoniae
Mycoplasma pneumoniae
Mycoplasma pneumoniae
Bordetella holmesii
Bordetella holmesii
Bordetella holmesii
Bordetella holmesii
Bordetella holmesii
Bordetella holmesii
Bordetella holmesii
Bordetella holmesii
Bordetella holmesii
Bordetella pertussis
Bordetella pertussis
Bordetella pertussis
Bordetella pertussis
Bordetella pertussis
Bordetella pertussis
Bordetella parapertussis
Bordetella parapertussis
Bordetella parapertussis
Bordetella bronchiosceptica
Bordetella bronchiosceptica
Bordetella bronchiosceptica
Bordetella bronchiosceptica
chlamydia pneumoniae
chlamydia pneumoniae
chlamydia pneumoniae
chlamydia pneumoniae
legionella pneumophila
legionella pneumophila
legionella pneumophila
mycoplasma pneumoniae
mycoplasma pneumoniae
mycoplasma pneumoniae
mycoplasma pneumoniae
mycoplasma pneumoniae
mammarenavirus L segment
mammarenavirus L segment
mammarenavirus L segment
mammarenavirus S segment
mammarenavirus S segment
mammarenavirus S segment
Lassa mammarenavirus
Lassa mammarenavirus
Lassa mammarenavirus
Argentinian mammarenavirus
Argentinian mammarenavirus
Argentinian mammarenavirus
Machupo mammarenavirus
Machupo mammarenavirus
Machupo mammarenavirus
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Staphylococcus aureus gyrA
Staphylococcus aureus gyrA
Staphylococcus aureus gyrA
Staphylococcus aureus gyrB
Staphylococcus aureus gyrB
Staphylococcus aureus gyrB
Staphylococcus aureus
thermonuclease
Staphylococcus aureus
thermonuclease
Staphylococcus aureus
thermonuclease
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Staphylococcus aureus gyrA
Staphylococcus aureus gyrA
Staphylococcus aureus gyrA
Staphylococcus aureus gyrB
Staphylococcus aureus gyrB
Staphylococcus aureus gyrB
Staphylococcus aureus
thermonuclease
Staphylococcus aureus
thermonuclease
Staphylococcus aureus
thermonuclease
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Acinetobacter spp. 16S-23S
Proteus mirabilis rpoD
Proteus mirabilis rpoD
Proteus mirabilis rpoD
Proteus vulgaris 16S
Proteus vulgaris 16S
Proteus vulgaris 16S
pitti gyrB
pitti gyrB
pitti gyrB
Enterobacter nimipressuralis
Enterobacter nimipressuralis
Enterobacter nimipressuralis
Bacillus anthracis pagA
Bacillus anthracis pagA
Bacillus anthracis pagA
Bacillus anthracis capB
Bacillus anthracis capB
Bacillus anthracis capB
Brucella spp. 23S
Brucella spp. 23S
Brucella spp. 23S
Brucella spp. bcsp31
Brucella spp. bcsp31
Brucella spp. bcsp31
Brucella spp. omp2a
Brucella spp. omp2a
Brucella spp. omp2a
Coxiella burnetii IS110
Coxiella burnetii IS110
Coxiella burnetii IS110
Francisella tularensis 16S
Francisella tularensis 16S
Francisella tularensis 16S
Rickettsia spp. 16S
Rickettsia spp. 16S
Rickettsia spp. 16S
Rickettsia spp. 23S
Rickettsia spp. 23S
Rickettsia spp. 23S
Rickettsia spp. 782 - 17K genus
Rickettsia spp. 782 - 17K genus
Rickettsia spp. 782 - 17K genus
Rickettsia spp. 783 - 17K genus
Rickettsia spp. 783 - 17K genus
Rickettsia spp. 783 - 17K genus
Yersinia pestis pMT1
Yersinia pestis pMT1
Yersinia pestis pMT1
Yersinia pestis pCD1
Yersinia pestis pCD1
Yersinia pestis pCD1
Yersinia pestis pPCP1
Yersinia pestis pPCP1
Yersinia pestis pPCP1
Acinetobacter spp.
Acinetobacter spp.
Acinetobacter spp.
A. pittii gyrB
A. pittii gyrB
A. pittii gyrB
A. calcoaceticus gyrB
A. calcoaceticus gyrB
A. calcoaceticus gyrB
Bacillus anthracis pagA
Bacillus anthracis pagA
Bacillus anthracis pagA
Bacillus anthracis capB
Bacillus anthracis capB
Bacillus anthracis capB
Francisella tularensis tul4
Francisella tularensis tul4
Francisella tularensis tul4
Francisella tularensis fopA
Francisella tularensis fopA
Francisella tularensis fopA
Yersinia pestis pMT1
Yersinia pestis pMT1
Yersinia pestis pMT1
Yersinia pestis pCD1
Yersinia pestis pCD1
Yersinia pestis pCD1
Yersinia pestis pPCP1
Yersinia pestis pPCP1
Yersinia pestis pPCP1
Coxiella burnetii com1
Coxiella burnetii com1
Coxiella burnetii com1
Coxiella burnetii IS110
Coxiella burnetii IS110
Coxiella burnetii IS110
Rickettsia spp. 782 - 17K genus
Rickettsia spp. 782 - 17K genus
Rickettsia spp. 782 - 17K genus
Rickettsia spp. 783 - 17K genus
Rickettsia spp. 783 - 17K genus
Rickettsia spp. 783 - 17K genus
Brucella spp. bcsp31
Brucella spp. bcsp31
Brucella spp. bcsp31
Brucella spp. omp2a
Brucella spp. omp2a
Brucella spp. omp2a
Proteus vulgaris rpoD
Proteus vulgaris rpoD
Proteus vulgaris rpoD
Proteus penneri rpoD
Proteus penneri rpoD
Proteus penneri rpoD
Proteus hauseri rpoD
Proteus hauseri rpoD
Proteus hauseri rpoD
baumannii gyrB
baumannii gyrB
baumannii gyrB
nosocomialis gyrB
nosocomialis gyrB
nosocomialis gyrB
Enterobacter cloacae dnaJ
Enterobacter cloacae dnaJ
Enterobacter cloacae dnaJ
Enterobacter asburiae pyrG
Enterobacter asburiae pyrG
Enterobacter asburiae pyrG
Enterobacter asburiae dnaJ
Enterobacter asburiae dnaJ
Enterobacter asburiae dnaJ
Enterobacter hormaechei pyrG
Enterobacter hormaechei pyrG
Enterobacter hormaechei pyrG
Enterobacter hormaechei dnaJ
Enterobacter hormaechei dnaJ
Enterobacter hormaechei dnaJ
Enterobacter kobei pyrG
Enterobacter kobei pyrG
Enterobacter kobei pyrG
Enterobacter kobei dnaJ
Enterobacter kobei dnaJ
Enterobacter kobei dnaJ
Enterobacter ludwigii pyrG
Enterobacter ludwigii pyrG
Enterobacter ludwigii pyrG
Enterobacter ludwigii dnaJ
Enterobacter ludwigii dnaJ
Enterobacter ludwigii dnaJ
Enterobacter mori pyrG
Enterobacter mori pyrG
Enterobacter mori pyrG
Enterobacter mori dnaJ
Enterobacter mori dnaJ
Enterobacter mori dnaJ
A. baumannii gyrB
A. baumannii gyrB
A. baumannii gyrB
A. nosocomialis gyrB
A. nosocomialis gyrB
A. nosocomialis gyrB
Enterobacter cloacae dnaJ
Enterobacter cloacae dnaJ
Enterobacter cloacae dnaJ
Enterobacter asburiae dnaJ
Enterobacter asburiae dnaJ
Enterobacter asburiae dnaJ
Enterobacter asburiae pyrG
Enterobacter asburiae pyrG
Enterobacter asburiae pyrG
Enterobacter hormaechei pyrG
Enterobacter hormaechei pyrG
Enterobacter hormaechei pyrG
Enterobacter hormaechei dnaJ
Enterobacter hormaechei dnaJ
Enterobacter hormaechei dnaJ
Enterobacter kobei pyrG
Enterobacter kobei pyrG
Enterobacter kobei pyrG
Enterobacter kobei dnaJ
Enterobacter kobei dnaJ
Enterobacter kobei dnaJ
Enterobacter ludwigii pyrG
Enterobacter ludwigii pyrG
Enterobacter ludwigii pyrG
Enterobacter ludwigii dnaJ
Enterobacter ludwigii dnaJ
Enterobacter ludwigii dnaJ
Enterobacter mori pyrG
Enterobacter mori pyrG
Enterobacter mori pyrG
Enterobacter mori dnaJ
Enterobacter mori dnaJ
Enterobacter mori dnaJ
Nocardia 16S
Nocardia 16S
Nocardia 16S
Nocardia hsp65
Nocardia hsp65
Nocardia hsp65
Nocardia gyrB
Nocardia gyrB
Nocardia gyrB
Nocardia secA1
Nocardia secA1
Nocardia secA1
Nocardia sodA
Nocardia sodA
Nocardia sodA
Cryptococcus 18S
Cryptococcus 18S
Cryptococcus 18S
Cryptococcus URA5
Cryptococcus URA5
Cryptococcus URA5
Cryptococcus ITS
Cryptococcus ITS
Cryptococcus ITS
Cryptococcus 28S
Cryptococcus 28S
Cryptococcus 28S
Cryptococcus CTX1
Cryptococcus CTX1
Cryptococcus CTX1
Actinomyces 16S
Actinomyces 16S
Actinomyces 16S
Nocardia 16S
Nocardia 16S
Nocardia 16S
Nocardia hsp65
Nocardia hsp65
Nocardia hsp65
Nocardia secA1
Nocardia secA1
Nocardia secA1
Nocardia sodA
Nocardia sodA
Nocardia sodA
Cryptococcus 18S
Cryptococcus 18S
Cryptococcus 18S
Cryptococcus URA5
Cryptococcus URA5
Cryptococcus URA5
Cryptococcus ITS
Cryptococcus ITS
Cryptococcus ITS
Cryptococcus 28S
Cryptococcus 28S
Cryptococcus 28S
Cryptococcus CTX1
Cryptococcus CTX1
Cryptococcus CTX1
Actinomyces 16S
Actinomyces 16S
Actinomyces 16S
Streptococcus 16S
Streptococcus 16S
Streptococcus 16S
Streptococcus tuf
Streptococcus tuf
Streptococcus tuf
Streptococcus sodA
Streptococcus sodA
Streptococcus sodA
Streptococcus rpoB
Streptococcus rpoB
Streptococcus rpoB
Aspergillus 18S
Aspergillus 18S
Aspergillus 18S
Aspergillus 28S
Aspergillus 28S
Aspergillus 28S
Aspergillus ITS
Aspergillus ITS
Aspergillus ITS
Candida ITS
Candida ITS
Candida ITS
Candida 28S
Candida 28S
Candida 28S
Candida Mitochondrion
Candida Mitochondrion
Candida Mitochondrion
Candida albicans ERG11
Candida albicans ERG11
Candida albicans ERG11
Candida lusitaniae ERG9
Candida lusitaniae ERG9
Candida lusitaniae ERG9
Clostridium difficile 16S
Clostridium difficile 16S
Clostridium difficile 16S
Clostridium perfringens 16S
Clostridium perfringens 16S
Clostridium perfringens 16S
Clostridium tetani 16S
Clostridium tetani 16S
Clostridium tetani 16S
Campylobacteraceae 16S
Campylobacteraceae 16S
Campylobacteraceae 16S
Campylobacteraceae 23S
Campylobacteraceae 23S
Campylobacteraceae 23S
Campylobacteraceae flaG
Campylobacteraceae flaG
Campylobacteraceae flaG
Corynebacterium 16S
Corynebacterium 16S
Corynebacterium 16S
Corynebacterium rpoB
Corynebacterium rpoB
Corynebacterium rpoB
Corynebacterium tox
Corynebacterium tox
Corynebacterium tox
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Corynebacterium
pseudotuberculosis chrA
Corynebacterium
pseudotuberculosis chrA
Corynebacterium
pseudotuberculosis chrA
Corynebacterium
pseudotuberculosis pld
Corynebacterium
pseudotuberculosis pld
Corynebacterium
pseudotuberculosis pld
Corynebacterium
striatum chrA
Corynebacterium
striatum chrA
Corynebacterium
striatum chrA
Corynebacterium ulcerans pld
Corynebacterium ulcerans pld
Corynebacterium ulcerans pld
Escherichia coli O157:H1
Escherichia coli O157:H1
Escherichia coli O157:H1
Escherichia coli O0157:H1
Escherichia coli O157:H1
Escherichia coli O157:H1
Listeria monocytogenes 16S
Listeria monocytogenes 16S
Listeria monocytogenes 16S
Listeria monocytogenes 23S
Listeria monocytogenes 23S
Listeria monocytogenes 23S
Listeria monocytogenes prfA
Listeria monocytogenes prfA
Listeria monocytogenes prfA
Mycobacterium bovis 16S
Mycobacterium bovis 16S
Mycobacterium bovis 16S
Neisseria meningitidis 16S
Neisseria meningitidis 16S
Neisseria meningitidis 16S
Neisseria meningitidis 23S
Neisseria meningitidis 23S
Neisseria meningitidis 23S
Salmonella enterica Paratyphi
Salmonella enterica Paratyphi
Salmonella enterica Paratyphi
Salmonella enterica Typhi
Salmonella enterica Typhi
Salmonella enterica Typhi
Salmonella enterica Typhi 16S
Salmonella enterica Typhi 16S
Salmonella enterica Typhi 16S
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Yersinia enterocolitica 16S
Yersinia enterocolitica 16S
Yersinia enterocolitica 16S
Yersinia enterocolitica 23S
Yersinia enterocolitica 23S
Yersinia enterocolitica 23S
Yersinia pseudotuberculosis
Yersinia pseudotuberculosis
Yersinia pseudotuberculosis
Vibrio cholerae 16S
Vibrio cholerae 16S
Vibrio cholerae 16S
Vibrio cholerae hlyA
Vibrio cholerae hlyA
Vibrio cholerae hly A
Vibrio parahaemolyticus 23S
Vibrio parahaemolyticus 23S
Vibrio parahaemolyticus 23S
Vibrio vulnificus 16S
Vibrio vulnificus 16S
Vibrio vulnificus 16S
Vibrio vulnificus 23S
Vibrio vulnificus 23S
Vibrio vulnificus 23S
Cronobacter flk
Cronobacter flk
Cronobacter flk
Mycoplasma capricolum
capripneumoniae 23S
Mycoplasma capricolum
capripneumoniae 23S
Mycoplasma capricolum
capripneumoniae 23S
Mycoplasma capricolum
capripneumoniae L22
Mycoplasma capricolum
capripneumoniae L22
Mycoplasma capricolum
capripneumoniae L22
Mycoplasma mycoides
mycoides 23S
Mycoplasma mycoides
mycoides 23S
Mycoplasma mycoides
mycoides 23S
Mycoplasma mycoides
mycoides lppB
Mycoplasma mycoides
mycoides lppB
Mycoplasma mycoides
mycoides lppB
Andes Orthohantavirus
Andes Orthohantavirus
Andes Orthohantavirus
Andes Orthohantavirus
Andes Orthohantavirus
Andes Orthohantavirus
Andes Orthohantavirus
Andes Orthohantavirus
Andes Orthohantavirus
Dobrava-Begrade
orthohantavirus Segment S
Dobrava-Begrade
orthohantavirus Segment S
Dobrava-Begrade
orthohantavirus Segment S
Dobrava-Begrade
orthohantavirus Segment M
Dobrava-Begrade
orthohantavirus Segment M
Dobrava-Begrade
orthohantavirus Segment M
Dobrava-Begrade
orthohantavirus Segment L
Dobrava-Begrade
orthohantavirus Segment L
Dobrava-Begrade
orthohantavirus Segment L
Hantaan orthohantavirus
Hantaan orthohantavirus
Hantaan orthohantavirus
Hantaan orthohantavirus
Hantaan orthohantavirus
Hantaan orthohantavirus
Hantaan orthohantavirus
Hantaan orthohantavirus
Hantaan orthohantavirus
Puumala orthohantavirus
Puumala orthohantavirus
Puumala orthohantavirus
Puumala orthohantavirus
Puumala orthohantavirus
Puumala orthohantavirus
Puumala orthohantavirus
Puumala orthohantavirus
Puumala orthohantavirus
Seoul orthohantavirus Segment
Seoul orthohantavirus Segment
Seoul orthohantavirus Segment
Seoul orthohantavirus Segment
Seoul orthohantavirus Segment
Seoul orthohantavirus Segment
Seoul orthohantavirus Segment
Seoul orthohantavirus Segment
Seoul orthohantavirus Segment
Nombre orthohantavirus
Nombre orthohantavirus
Nombre orthohantavirus
Nombre orthohantavirus
Nombre orthohantavirus
Nombre orthohantavirus
Nombre orthohantavirus
Nombre orthohantavirus
Nombre orthohantavirus
Mycobacterium bovis 23S
Mycobacterium bovis 23S
Mycobacterium bovis 23S
Escherichia coli 23S
Escherichia coli 23S
Escherichia coli 23S
Cryptococcus neoformans
Cryptococcus neoformans
Cryptococcus neoformans
Aspergillus 18S
Aspergillus 18S
Aspergillus 18S
Aspergillus 28S
Aspergillus 28S
Aspergillus 28S
Aspergillus ITS
Aspergillus ITS
Aspergillus ITS
Aspergillus flavus
Aspergillus flavus
Aspergillus flavus
Aspergillus fumigatus
Aspergillus fumigatus
Aspergillus fumigatus
Candida ITS
Candida ITS
Candida ITS
Candida 18S
Candida 18S
Candida 18S
Candida 28S
Candida 28S
Candida 28S
Candida Mitochondrion
Candida Mitochondrion
Candida Mitochondrion
Candida albicans ERG11
Candida albicans ERG11
Candida albicans ERG11
Candida lusitaniae ERG9
Candida lusitaniae ERG9
Candida lusitaniae ERG9
Clostridium difficile 16S
Clostridium difficile 16S
Clostridium difficile 16S
Clostridium perfringens 16S
Clostridium perfringens 16S
Clostridium perfringens 16S
Clostridium tetani 16S
Clostridium tetani 16S
Clostridium tetani 16S
Corynebacterium 16S
Corynebacterium 16S
Corynebacterium 16S
Corynebacterium rpoB
Corynebacterium rpoB
Corynebacterium rpoB
Corynebacterium tox
Corynebacterium tox
Corynebacterium tox
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Corynebacterium
pseudotuberculosis pld
Corynebacterium
pseudotuberculosis pld
Corynebacterium
pseudotuberculosis pld
Corynebacterium
striatum chrA
Corynebacterium
striatum chrA
Corynebacterium
striatum chrA
Corynebacterium ulcerans pld
Corynebacterium ulcerans pld
Corynebacterium ulcerans pld
Campylobacteraceae 16S
Campylobacteraceae 16S
Campylobacteraceae 16S
Campylobacteraceae 23S
Campylobacteraceae 23S
Campylobacteraceae 23S
Campylobacteraceae flaG
Campylobacteraceae flaG
Campylobacteraceae flaG
Escherichia coli 23S
Escherichia coli 23S
Escherichia coli 23S
Escherichia coli O157:H1
Escherichia coli O157:H1
Escherichia coli O157:H1
Escherichia coli O157:H1
Escherichia coli O157:H1
Escherichia coli O157:H1
Shigella Virulence Factor
Shigella Virulence Factor
Shigella Virulence Factor
Shigella Virulence Factor
Shigella Virulence Factor
Shigella Virulence Factor
Listeria monocytogenes 16S
Listeria monocytogenes 16S
Listeria monocytogenes 16S
Listeria monocytogenes 23S
Listeria monocytogenes 23S
Listeria monocytogenes 23S
Listeria monocytogenes prfA
Listeria monocytogenes prfA
Listeria monocytogenes prfA
Mycobacterium bovis 16S
Mycobacterium bovis 16S
Mycobacterium bovis 16S
Mycobacterium bovis 23S
Mycobacterium bovis 23S
Mycobacterium bovis 23S
Neisseria meningitidis 16S
Neisseria meningitidis 16S
Neisseria meningitidis 16S
Neisseria meningitidis 23S
Neisseria meningitidis 23S
Neisseria meningitidis 23S
Salmonella enterica Paratyphi
Salmonella enterica Paratyphi
Salmonella enterica Paratyphi
Salmonella enterica Paratyphi
Salmonella enterica Paratyphi
Salmonella enterica Paratyphi
Salmonella enterica Typhi
Salmonella enterica Typhi
Salmonella enterica Typhi
Salmonella enterica Typhi
Salmonella enterica Typhi
Salmonella enterica Typhi
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Salmonella enterica Enteritides
Yersinia enterocolitica 16S
Yersinia enterocolitica 16S
Yersinia enterocolitica 16S
Yersinia enterocolitica 23S
Yersinia enterocolitica 23S
Yersinia enterocolitica 23S
Yersinia pseudotuberculosis
Yersinia pseudotuberculosis
Vibrio cholerae 16S
Vibrio cholerae 16S
Vibrio cholerae 16S
Vibrio cholerae hlyA
Vibrio cholerae hlyA
Vibrio cholerae hlyA
Vibrio parahaemolyticus 16S
Vibrio parahaemolyticus 16S
Vibrio parahaemolyticus 16S
Vibrio vulnificus 16S
Vibrio vulnificus 16S
Vibrio vulnificus 16S
Vibrio vulnificus 23S
Vibrio vulnificus 23S
Vibrio vulnificus 23S
Mycoplasma capricolum
capripneumoniae 23S
Mycoplasma capricolum
capripneumoniae 23S
Mycoplasma capricolum
capripneumoniae 23S
Mycoplasma capricolum
capripneumoniae 16S
Mycoplasma capricolum
capripneumoniae 16S
Mycoplasma capricolum
capripneumoniae 16S
Mycoplasma capricolum
capripneumoniae L22
Mycoplasma capricolum
capripneumoniae L22
Mycoplasma capricolum
capripneumoniae L22
Mycoplasma mycoides
mycoides 23S
Mycoplasma mycoides
mycoides 23S
Mycoplasma mycoides
mycoides 23S
Mycoplasma mycoides
mycoides 16S
Mycoplasma mycoides
mycoides 16S
Mycoplasma mycoides
mycoides 16S
Mycoplasma mycoides
mycoides lppB
Mycoplasma mycoides
mycoides lppB
Mycoplasma mycoides
mycoides lppB
Enterobacter cloacae
Enterobacter cloacae
Enterobacter cloacae
Klebsiella oxytoca
Klebsiella oxytoca
Klebsiella oxytoca
Klebsiella pneumoniae
Klebsiella pneumoniae
Klebsiella pneumoniae
Cryptococcus neoformans
Cryptococcus neoformans
Cryptococcus neoformans
Corynebacterium
pseudotuberculosis chrA
Corynebacterium
pseudotuberculosis chrA
Corynebacterium
pseudotuberculosis chrA
Escherichia coli 16S
Escherichia coli 16S
Escherichia coli 16S
Klebsiella variicola
Klebsiella variicola
Klebsiella variicola
Vibrio parahaemolyticus 23S
Vibrio parahaemolyticus 23S
Vibrio parahaemolyticus 23S
Reporters, which can be referred to interchangeably reporters, or detector nucleic acids, described herein are compatible for use in the devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, and lateral flow devices) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample (e.g., DETECTR reactions). Described herein is a reporter comprising a single stranded detector nucleic acid comprising a detection moiety, wherein the reporter is capable of being cleaved by the activated programmable nuclease, thereby generating a first detectable signal. As used herein, a detector nucleic acid is used interchangeably with reporter or reporter. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the detector nucleic acid is a single-stranded nucleic acid comprising ribonucleotides. The detector nucleic acid can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the detector nucleic acid may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some cases, the detector nucleic acid has only ribonucleotide residues. In some cases, the detector nucleic acid has only deoxyribonucleotide residues. In some cases, the detector nucleic acid may comprise nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the detector nucleic acid may comprise synthetic nucleotides. In some cases, the detector nucleic acid may comprise at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the detector nucleic acid is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length. In some cases, the detector nucleic acid is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length. In some cases, the detector nucleic acid may comprise at least one uracil ribonucleotide. In some cases, the detector nucleic acid may comprise at least two uracil ribonucleotides. Sometimes the detector nucleic acid has only uracil ribonucleotides. In some cases, the detector nucleic acid may comprise at least one adenine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two adenine ribonucleotide. In some cases, the detector nucleic acid has only adenine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one cytosine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two cytosine ribonucleotide. In some cases, the detector nucleic acid may comprise at least one guanine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two guanine ribonucleotide. A detector nucleic acid can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the detector nucleic acid is from 5 to 12 nucleotides in length. In some cases, the detector nucleic acid is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the detector nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. For cleavage by a programmable nuclease comprising Cas13, a detector nucleic acid can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Cas12, a detector nucleic acid can be 10 nucleotides in length.
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 detector nucleic acid by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, a signal may be present in the absence of detector nucleic acid cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal may be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.
The reagents described herein can also include buffers, which are compatible with the devices, systems, fluidic devices, kits, and methods disclosed herein. The buffers described herein are compatible for use in the devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, and lateral flow devices) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether the target nucleic acid is in the sample (e.g., DETECTR reactions). These buffers are compatible with the other reagents, samples, and support mediums as described herein for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry. The methods described herein can also include the use of buffers, which are compatible with the methods disclosed herein. For example, a buffer may comprise HEPES, MES, TCEP, EGTA, Tween 20, KCl, MgCl2, glycerol, or any combination thereof. In some instances, a buffer may comprise Tris-HCl pH 8.8, VLB, EGTA, CH3COOH, TCEP, IsoAmp®, (NH4)2SO4, KCl, MgSO4, Tween20, KOAc, MgOAc, BSA, TCEP, or any combination thereof. In some instances the buffer may comprise from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM HEPES pH 6.8. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KCl. In other instances the buffer may comprise 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl2. The buffer can comprise 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30% glycerol. The buffer can comprise from 0% to 30%, from 5% to 30%, from 10% to 30%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 0% to 25%, from 2% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, from 0% to 20%, from 5% to 20%, from 10% to 20%, from 15% to 20%, from 0% to 15%, from 5% to 15%, from 10% to 15%, from 0% to 10%, from 5% to 10%, or from 0% to 5% glycerol. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM Tris-HCl pH 8.8. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KOAc. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM MgOAc. In some instances the buffer may comprise from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM EGTA. The buffer can comprise from 0% to 30%, from 5% to 30%, from 10% to 30%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 0% to 25%, from 2% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, from 0% to 20%, from 5% to 20%, from 10% to 20%, from 15% to 20%, from 0% to 15%, from 5% to 15%, from 10% to 15%, from 0% to 10%, from 5% to 10%, or from 0% to 5% Tween 20.
In some embodiments, the reagents described herein may include a composition for improving detection signal strength, detection reaction time, detection reaction efficiency, stability, solubility, or the like. In some embodiments, the composition may comprise one or more additives. The one or more additives may, for example, comprise amino acids or derivatives thereof, chaotrpes, chelators, cyclodextrins, inhibitors, ionic liquids, linkers, metals, non detergent sulfobetaines, organic acids, osmolytes, peptides, polyamides, polymers, polyols, polyols and salts, salts, or combinations thereof. In some embodiments, the one or more additives may, for example, comprise one or more of trichloroacetic acid, L-Arginine, L-Glutamic acid, glycine, L-Proline, L-Histidine, beta (B)-Alanine, L-Serine, L-Arginine ethyl ester dihydrochloride, L-Argininamide dihydrochloride, 6-Aminohexanoic acid, Gly-gly peptide, Gly-gly-gly peptide, tryptone, betaine monohydrate, D-(+)-Trehalose dihydrate, Xylitol, D-Sorbitol, sucrose, hydroxyectoine, Trimethylamine N-oxide dihydrate, methyl alpha (α)-D-gluocopyranoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, adipic acid, ethylenediamine dihydrochloride, guanidine hydrochloride, urea, N-methylurea, N-ethylurea, N-methylformamide, hypotauring, TCEP hydrochloride, GSH (L-Glutathione reduced), GSSG (L-Glutathione oxidized), benzaminidine hydrochloride, ethylenediaminetetraacetic acid disodium salt dihydrate, magnesium chloride hexahydrate, calcium chloride dihydrate, cadmium chloride hydrate, cobalt (II) chloride hexahydrate, Non Detergent Sulfobetaine 195 (NDSB-195), NDSB-201, NDSB-211, NDSB-221, NDSB-256, taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, tacsimate pH 7.0, tetraethylammonium bromide, cholin acetate, 1-Ethyl-3-methylimidazolium acetate, 1-Butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, potassium thiocynate, gadolinium (III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium nitrate, DL-malic acid pH 7.0, lithium citrate tribasic tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p-toluenesulfonate, sodium chloride, potassium chloride, sodium phosphate monobasic monohydrate, potassium phosphate dibasic, sodium sulfate decahydrate, lithium chloride, sodium bromide, glycerol, ethylene glycol, polyethylene glycol 200 (PEG-200), PEG 3350, PEG 8000, PEG monomethyl ether 550, PEG monomethyl ether 750, PEG monomethyl ether 1900, formamide, polypropylene glycol P 400, pentaerythritol ethoxylate, 1,2-Propanediol, polyvinylpyrrolidone K 15, 6-O-α-Maltosyl-β-cyclodextrin, (2-Hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, Methyl-β-cyclodextrin, or any combination thereof.
In some embodiments, the reagents described herein may include a composition for increasing the signal strength of any of the assays described herein. In some embodiments, the composition comprises water and an additive. In some embodiments, the additive may comprise trehalose, xylitol, D-sorbitol, sucrose, and trimethylamine N-oxide dihydrate, or any combination thereof. In some embodiments, the composition further comprises one or more targets, one or more enzymes, one or more reporters, one or more substrates, or any combination thereof as described herein.
In some embodiments, the reagents described herein may include a composition for reducing the time it takes for the signal of any of the assays described herein to saturate. In some embodiments, the composition comprises an additive. In some embodiments, the additive may comprise betaine monohydrate, acetamide, GABA, L-proline, beta-alanine, 6-aminohexanonic acid, urea, methylurea, ethylurea, hypotaurine, NDSB-256, ammonium acetate, or any combination thereof. In some embodiments, the composition further comprises one or more targets, one or more enzymes, one or more reporters, one or more substrates, or any combination thereof as described herein.
In some embodiments, the additive is present at a concentration of at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, at least about 10 μM, at least about 20 μM, at least about 30 μM, at least about 40 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 200 μM, at least about 300 μM, at least about 400 μM, at least about 500 μM, at least about 600 μM, at least about 700 μM, at least about 800 μM, at least about 900 μM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 200 mM, at least about 300 mM, at least about 400 mM, at least about 500 mM, at least about 600 mM, at least about 700 mM, at least about 800 mM, at least about 900 mM, at least about 1 M, at least about 2 M, at least about 3 M, at least about 4 M, at least about 5 M, at least about 6 M, at least about 7 M, at least about 8 M, at least about 9 M, or at least about 10 M.
In some embodiments, the composition increases the signal strength by at least about a factor of 1.01, at least about a factor of 1.02, at least about a factor of 1.03, at least about a factor of 1.04, at least about a factor of 1.05, at least about a factor of 1.06, at least about a factor of 1.07, at least about a factor of 1.08, at least about a factor of 1.09, at least about a factor of 1.1, at least about a factor of 1.2, at least about a factor of 1.3, at least about a factor of 1.4, at least about a factor of 1.5, at least about a factor of 1.6, at least about a factor of 1.7, at least about a factor of 1.8, at least about a factor of 1.9, at least about a factor of 2, at least about a factor of 3, at least about a factor of 4, at least about a factor of 5, at least about a factor of 6, at least about a factor of 7, at least about a factor of 8, at least about a factor of 9, at least about a factor of 10, at least about a factor of 20, at least about a factor of 30, at least about a factor of 40, at least about a factor of 50, at least about a factor of 60, at least about a factor of 70, at least about a factor of 80, at least about a factor of 90, at least about a factor of 100, at least about a factor of 200, at least about a factor of 300, at least about a factor of 400, at least about a factor of 500, at least about a factor of 600, at least about a factor of 700, at least about a factor of 800, at least about a factor of 900, or at least about a factor of 1000.
In some embodiments, the composition reduces the time it takes for the signal to saturate by at least about a factor of 1.01, at least about a factor of 1.02, at least about a factor of 1.03, at least about a factor of 1.04, at least about a factor of 1.05, at least about a factor of 1.06, at least about a factor of 1.07, at least about a factor of 1.08, at least about a factor of 1.09, at least about a factor of 1.1, at least about a factor of 1.2, at least about a factor of 1.3, at least about a factor of 1.4, at least about a factor of 1.5, at least about a factor of 1.6, at least about a factor of 1.7, at least about a factor of 1.8, at least about a factor of 1.9, at least about a factor of 2, at least about a factor of 3, at least about a factor of 4, at least about a factor of 5, at least about a factor of 6, at least about a factor of 7, at least about a factor of 8, at least about a factor of 9, at least about a factor of 10, at least about a factor of 20, at least about a factor of 30, at least about a factor of 40, at least about a factor of 50, at least about a factor of 60, at least about a factor of 70, at least about a factor of 80, at least about a factor of 90, at least about a factor of 100, at least about a factor of 200, at least about a factor of 300, at least about a factor of 400, at least about a factor of 500, at least about a factor of 600, at least about a factor of 700, at least about a factor of 800, at least about a factor of 900, or at least about a factor of 1000. In some embodiments, the additive is present at a concentration of at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, at least about 10 μM, at least about 20 μM, at least about 30 μM, at least about 40 μM, at least about 50 μM, at least about 60 M, at least about 70 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 200 μM, at least about 300 μM, at least about 400 μM, at least about 500 μM, at least about 600 μM, at least about 700 μM, at least about 800 μM, at least about 900 μM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 200 mM, at least about 300 mM, at least about 400 mM, at least about 500 mM, at least about 600 mM, at least about 700 mM, at least about 800 mM, at least about 900 mM, at least about 1 M, at least about 2 M, at least about 3 M, at least about 4 M, at least about 5 M, at least about 6 M, at least about 7 M, at least about 8 M, at least about 9 M, at least about 10 M.
In some embodiments, the one or more enzymes may comprise a nuclease. In some embodiments, the one or more enzymes may comprise a programmable nuclease. In some embodiments, the one or more enzymes may comprise a Cas12 enzyme. In some embodiments, the one or more enzymes may comprise a Cas14 enzyme. In some embodiments, the one or more enzymes may comprise a CasPhi enzyme. In some embodiments, the one or more enzymes may comprise a Cas13 enzyme. In some embodiments, the one or more enzymes may comprise HRP. In some embodiments, the one or more enzymes may comprise any one or combination of enzymes presented in this disclosure.
In some embodiments, the one or more reporters may comprise a reporter free in solution. In some embodiments, the one or more reporters may comprise a reporter immobilized on a surface of a support. In some embodiments, the one or more reporters may comprise biotin. In some embodiments, the one or more reporters may comprise a fluorescent moiety. In some embodiments, the one or more reporters may comprise a nucleic acid tether. In some embodiments, the one or more reporters may comprise a linker. In some embodiments, the one or more reporters may comprise any one or combination of reporters presented in this disclosure.
In some embodiments, the one or more supports may comprise a glassy substance. In some embodiments, the one or more substrates may comprise a polymeric substance. In some embodiments, the one or more substrates may comprise a hydrogel. In some embodiments, the one or more substrates may comprise any one or combination of substrates presented in this disclosure.
In some embodiments, a sliding valve device may comprise a first chamber for sample lysis, a second chamber for detection, and a third chamber for amplification. Another way of referring to these chambers is a sample chamber (e.g., the first chamber), a detection chamber (e.g., the second chamber), and an amplification chamber (e.g., the third chamber). In this layout, the present disclosure provides a device for measuring a signal which may comprise: a sliding layer comprising a channel with an opening at a first end of the channel and an opening at a second end of the channel; and a fixed layer comprising: i) a first chamber having an opening; ii) a second chamber having an opening, wherein the second chamber may comprise a programmable nuclease and a reporter comprising a nucleic acid and a detection moiety; iii) a first side channel having an opening aligned with the opening of the first chamber; and iv) a second side channel having an opening aligned with the opening of the second chamber, wherein the sliding layer and the fixed layer move relative to each other to fluidically connect the first chamber and the first side channel via the opening at the first end of the channel, the opening at the second end of the channel, the opening of the first chamber, and the opening of the first side channel, and wherein the sliding layer and the fixed layer move relative to each other to fluidically connect the second chamber and the second side channel via the opening at the first end of the channel, the opening at the second end of the channel, the opening of the second chamber, and the opening of the second side channel. The fixed layer further may comprise i) a third chamber having an opening; and ii) a third side channel having an opening aligned with the opening of the third chamber, wherein the sliding layer and the fixed layer move relative to each other to fluidically connect the third chamber and the third side channel via the opening at the first end of the channel, the opening at the second end of the channel, the opening of the third chamber, and the opening of the third side channel. The second chamber is coupled to a measurement device for measuring the signal from the detection moiety produced by cleavage of the nucleic acid of the reporter. Additionally, the opening of the first end of the channel overlaps with the opening of the first chamber and the opening of the second end of the channel overlaps with the opening of the first side channel. The opening of the first end of the channel overlaps with the opening of the second chamber and the opening of the second end of the channel overlaps with the opening of the second side channel. The opening of the first end of the channel overlaps with the opening of the third chamber and the opening of the second end of the channel overlaps with the opening of the third channel. Additionally, the first side channel, the second side channel, and the third side channel are fluidically connected to a mixing chamber. In this embodiment, the second chamber additionally includes a guide nucleic acid.
In another embodiment, a sliding valve device may comprise a first chamber for sample lysis and a second chamber for detection. Another way of referring to these chambers is a sample chamber (e.g., the first chamber) and a detection chamber. In this layout, the present disclosure provides a device for measuring a signal may comprise: a sliding layer comprising a channel with an opening at a first end of the channel and an opening at a second end of the channel; and a fixed layer comprising: i) a first chamber having an opening; ii) a second chamber having an opening, wherein the second chamber may comprise a programmable nuclease and a reporter comprising a nucleic acid and a detection moiety; iii) a first side channel having an opening aligned with the opening of the first chamber; and iv) a second side channel having an opening aligned with the opening of the second chamber, wherein the sliding layer and the fixed layer move relative to each other to fluidically connect the first chamber and the first side channel via the opening at the first end of the channel, the opening at the second end of the channel, the opening of the first chamber, and the opening of the first side channel, and wherein the sliding layer and the fixed layer move relative to each other to fluidically connect the second chamber and the second side channel via the opening at the first end of the channel, the opening at the second end of the channel, the opening of the second chamber, and the opening of the second side channel. The second chamber is coupled to a measurement device for measuring the signal from the detection moiety produced by cleavage of the nucleic acid of the reporter. Additionally, the opening of the first end of the channel overlaps with the opening of the first chamber and the opening of the second end of the channel overlaps with the opening of the first side channel. The opening of the first end of the channel overlaps with the opening of the second chamber and the opening of the second end of the channel overlaps with the opening of the second side channel. Additionally, the first side channel and the second side channel are fluidically connected to a mixing chamber. In this embodiment, the second chamber additionally includes a guide nucleic acid.
In some embodiments, the DETECTR assay relies on fluorescence-based detection. In certain embodiments, the DETECTR assay relies on electrochemical-based detection. Electrochemical-based assays have been found to have a lower limit of detection than fluorescence-based assays by roughly two orders of magnitude (Lou et al., 2015).
In some embodiments, electrochemical probes are incorporated into the DETECTR assay to achieve a lower limit of detection. For example, the following electrochemical probe may comprise: 5′-2XXTTATTXX-3′, where 2=5′ 6-FAM; X=ferrocene dT; and 3′=3′ Biotin TEG, where TEG is a 15 atom triethylene glycol spacer. In some embodiments, electrochemical probes are tested with cyclic voltammetry. In some embodiments, electrochemical probes are tested with square wave voltammetry. In some embodiments, a DropSens μSTAT ECL instrument is used for electrochemical measurements. In some embodiments, DropSens screen-printed carbon electrodes are used for electrochemical measurements.
Potential (V) is shown on the x-axis from −0.4 V to 0.6 V in increments of 0.2 V. Current (μA) is shown on the y-axis from −1 μA to 1 μA in increments of 0.2 μA. The dark green line (1501) and light green line (1502) depict cyclic voltammograms taken before and after HERC2 DETECTR reaction using 24 μM electrochemical reporter, respectively. In this example, each trace is the average of three scans of the same solution and error bars represent standard deviations. The difference in current between the voltammograms indicates detection of HERC2 DETECTR at 24 μM.
In certain embodiments, described herein DETECTR has been demonstrated to be a powerful technology for detection of pathogens such as SARS-CoV-2 (Broughton et al., 2020). In some embodiments, electrochemical probes are utilized in the DETECTR assay for detection of pathogens. In some embodiments, the electrochemical probe-based DETECTR assay is configured for detection of the pathogen SARS-CoV-2. In some embodiments, the electrochemical probe is 5′-2XXTTATTXX-3′, where 2=5′ 6-FAM; X=ferrocene dT; and 3=3′ BiotinTEG.
CRISPR diagnostic reactions are generally performed in solution where the Cas protein-RNA complexes can freely bind target nucleic acids and reporters. However, reactions where all components are in solution limit the designs of CRISPR diagnostic assays, especially in microfluidic devices. A system where various components of the CRISPR diagnostic reaction are immobilized on a surface enables designs where multiple readouts can be accomplished within a single reaction chamber.
Described herein are various methods to immobilize CRISPR diagnostic reaction components to the surface of a reaction chamber or other surface (e.g., a surface of a bead). Any of the devices described herein may comprise one or more immobilized detection reagent components (e.g., programmable nuclease, guide nucleic acid, and/or reporter), or component capable of being immobilized at some point during a particular assay (e.g., through the inclusion of a functionality for immobilization to a surface, such as the surface of a bead). In some embodiments, the surface is a bead, which may be present at the beginning of a reaction, or added later to capture one or more components of a reaction (e.g., unreacted reporters comprising an enzyme, a linker, and a functionality for immobilization). The term “bead” is not limited to any particular size or shape. Beads may be uniform or non-uniform, spherical or non-spherical, regular or irregular. In embodiments, beads are magnetic or paramagnetic. The surface of the beads may comprise a reaction partner for the functional moiety for immobilization (e.g., streptavidin coated beads for capture of targets comprising a biotin functionality). In some embodiments, the beads are microparticles or nanoparticles. A variety of beads for use with nucleic acid based assays are commercially available, a non-limiting example of which includes DYNABEADS in various formats. In certain instances, methods include immobilization of programmable nucleases (e.g., Cas proteins or Cas enzymes), reporters, guide nucleic acids (e.g., gRNAs), or a combination of two or all of these. In some embodiments, various CRISPR diagnostic reaction components are modified with biotin. In some embodiments, these biotinylated CRISPR diagnostic reaction components are tested for immobilization on surfaces coated with streptavidin. In some embodiments, the biotin-streptavidin interaction is used as a model system for other immobilization chemistries.
Table 3 presents gRNA and reporter immobilization sequences
For some embodiments, described herein,
In some embodiments, the programmable nuclease, guide nucleic acid, or the reporter are immobilized to a device surface by a linkage or linker. In some embodiments, the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, a bond between streptavidin and biotin, an amide bond, or any combination thereof. In some embodiments, the linkage comprises non-specific absorption. In some embodiments, the programmable nuclease is immobilized to the device surface by the linkage, wherein the linkage is between the programmable nuclease and the surface. In some embodiments, the reporter is immobilized to the device surface by the linkage, wherein the linkage is between the reporter and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 5′ end of the guide nucleic acid and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 3′ end of the guide nucleic acid and the surface.
In some embodiments, various chemical modifications to gRNAs are described as shown in
In some embodiments, the immobilization of gRNAs to a streptavidin surface are described as shown in
In some embodiments, Cas proteins are complexed with RNA complexes as described herein and shown in
In some embodiments, reporters are immobilized to the surface as shown in
In some embodiments, combined RNP and a reporter system are immobilized for functional testing as shown in
In some embodiments, different reporters are immobilized in combination with Cas complexes on a streptavidin surface for evaluation of the DETECTR assay.
In some embodiments, Cy5 dye may be used as a reporter or a component of a reporter.
In some embodiments, gRNAs are modified. In some embodiments, the modified gRNAs are modified with linker molecules for immobilization onto a surface.
In some embodiments, guide RNAs are modified for surface modification. In some embodiments, reporters are modified for surface immobilization. In these embodiments, an immobilized gRNA, or immobilized reporter or a combination thereof participate in a diagnostic assay including a programmable nuclease.
In some embodiments, biomolecules are immobilized to surfaces. In some embodiments, the surfaces were glass.
Experimental parameters for the preparation of an embodiment of a complexing mix are seen in
In some embodiments, fluorescent quencher-based reporters are used in the immobilized DETECTR assay.
Described herein are various embodiments, where both the gRNA and reporter are bound to a plate as opposed to the gRNA, reporter and CAS protein. This removes the need to functionalize the surface with the pre-complex of gRNA and CAS protein, allowing for an easier manufacturing process. Additionally, greater specificity can be achieved by allowing for more stringent washes. An experimental design of this embodiment and conditions for binding a reporter to a plate in this embodiment are seen in
Described herein are various embodiments, that demonstrate target discrimination for immobilized reporters for the DETECTR reaction. An experiment design for such an embodiment is shown in
In some embodiments, a pneumatic pump interfaces with the cartridge. In some embodiments, as shown in a top down view, in the middle of
In some embodiments, the DETECTR assay device may comprise a sliding valve as seen in
In some embodiments, NHS-Amine chemistry is used for immobilization of DETECTR components.
In some embodiments, different combinations of reporter+guide+a Cas12 variant (SEQ ID NO: 17) are immobilized.
The results for optimizing gRNA and target concentrations to improve single-to-noise ratio for immobilized DETECTR assay are shown in
In some embodiments, amino modifications are used for DETECTR immobilization.
In some embodiments, rapid thermocycling and CRISPR diagnostics are used to detect SARS-CoV-2. Results are shown in
The top enzymes and buffers identified previously at various concentrations and with multiple replicates were tested for the FASTR assay. In some embodiments, the best performing enzymes and buffers as identified in the previously disclosed screening studies were used. Results of such embodiments are shown in
For some embodiments, single copy detection of SARS-CoV-2 with FASTR assay has been demonstrated as shown in
Variations on Across some embodiments, rapid cycling times are varied to evaluate denaturation and annealing/extension for the FASTR assay. Results for such embodiments are shown in
For some embodiments, the FASTR assay compatibility with crude lysis buffers was investigated. Results are shown in
For some embodiments non-optimized multiplexing of FASTR was demonstrated as shown in
In various aspects described herein, FASTR can be used for multiplexed detection, as shown in
In some embodiments, the FASTR assay enables multiplexed detection. Results of a limit of detection (LOD) study of such embodiments are shown in
In some embodiments, key primers and gRNAs have the sequences as listed in
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. 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.
Described herein are various embodiments of the DETECTR reaction involving optimization of sample preparation and lyophilization. Such embodiments allow for adapting the buffer for binding a substrate to perform a concentration step. In some embodiments, experiments may be performed to evaluate the lysis (sample is evaluated directly in the assay) and binding (the sample is eluted from magnetic beads) characteristics of buffers with different components. In such embodiments, the input sample is the same concentration as the eluted sample. Results showing strong lysis activity, but minimal binding/concentration potential are shown in
In some embodiments, crude lysis buffer is used in a one-pot assay with Cas14a.1. Results can be seen in
In some embodiments, the enzyme Cas12 variant (SEQ ID NO: 17) is used with the DETECR assay. In some embodiments, the sequence of the Cas12 variant is: MKKIDNFVGCYPVSKTLRFKAIPIGKTQENIEKKRLVEEDEVRAKDYKAVKKLIDRYHR EFIEGVLDNVKLDGLEEYYMLFNKSDREESDNKKIEIMEERFRRVISKSFKNNEEYKKIFS KKIIEEILPNYIKDEEEKELVKGFKGFYTAFVGYAQNRENMYSDEKKSTAISYRIVNENM PRFITNIK VFEKAKSILDVDKINEINEYILNNDYYVDDFFNIDFFNYVLNQKGIDIYNAIIG GIVTGDGRKIQGLNECINLYNQENKKIRLPQFKPLYKQILSESESMSFYIDEIESDDMLID MLKESLQIDSTINNAIDDLKVLFNNIFDYDLSGIFINNGLPITTISNDVYGQWSTISDGWNE RYDVLSNAKDKESEKYFEKRRKEYKKVKSFSISDLQELGGKDLSICKKINEIISEMIDDYK SKIEEIQYLFDIKELEKPLVTDLNKIELIKNSLDGLKRIERYVIPFLGTGKEQNRDEVFYGY FIKCIDAIKEIDGVYNKTRNYLTKKPYSKDKFKLYFENPQLMGGWDRNKESDYRSTLLR KNGKYYVAIIDKSSSNCMMNIEEDENDNYEKINYKLLPGPNKMLPKVFFSKKNREYFAP SKEIERIYSTGTFKKDTNFVKKDCENLITFYKDSLDRHEDWSKSFDFSFKESSAYRDISEF YRDVEKQGYRVSFDLLSSNAVNTL VEEGKLYLFQLYNKDFSEKSHGIPNLHTMYFRSLF DDNNKGNIRLNGGAEMFMRRASLNKQDVTVHKANQPIKNKNLLNPKKTTTLPYDVYK DKRFTEDQYEVHIPITMNKVPNNPYKINHMVREQLVKDDNPYVIGIDRGERNLIYVVVV DGQGHIVEQLSLNEIINENNGISIRTDYHTLLDAKERERDESRKQWKQIENIKELKEGYIS QVVHKICEL VEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLITKLNYMVDKKK DYNKPGGVLNGYQLTTQFESFSKMGTQNGIMFYIPAWLTSKMDPTTGFVDLLKPKYKN KADAQKFFSQFDSIRYDNQEDAFVFKVNYTKFPRTDADYNKEWEIYTNGERIRVFRNPK KNNEYDYETVNVSERMKELFDSYDLLYDKGELKETICEMEESKFFEELIKLFRLTLQMR NSISGRTDVDYLISPVKNSNGYFYNSNDYKKEGAKYPKDADANGAYNIARKVLWAIEQ FKMADEDKLDKTKISIKNQEWLEYAQTHCE* (SEQ ID NO: 17). The * indicates a stop codon.
For some embodiments, two groups of conditions were evaluated for lyophilization performance. For one embodiment, Group I, Trehalose is used.
In another embodiment, Group II comprising: PVP 40; sorbitol; Mannitol; and Mannosse are used.
In some embodiments, Trehalose is used to control the rate of the reaction.
Described herein are various methods and devices for carrying out DETECTR assays. In some embodiments, DETECTR assays utilize the Cas12 variant (SEQ ID NO: 17) protein. In some embodiments, RT-LAMP and DETECTR master mixes of reagents are lyophilized in the same combined master mix. In some embodiments, RT-LAMP and DETECTR master mixes of reagents and target are lyophilized in the same combined master mix.
In some embodiments, the reagents and target from both the RT-LAMP and DETECTR assays were lyophilized in one sample.
In some embodiments, the sample mixtures include multiple target nucleic acids. In some embodiments, the sample mixtures contain multiple copies of nucleic acid targets. In some embodiments, the 1000 copies (cps) of the nucleic acid target are present in one sample. In the legends on the right-hand sides of
In some embodiments, a master mix of assay reagents are reconstituted after lyophilization. In some embodiments, a master mix of DETECTR assay reagents are reconstituted after lyophilization. In some embodiments, a master mix of DETECTR assay reagents, including a Cas 12 variant (SEQ ID NO: 17) protein are reconstituted after lyophilization. In some embodiments, a master mix of amplification and DETECTR assay reagents, including Cas12 variant (SEQ ID NO: 17) protein are reconstituted after lyophilization. In some embodiments, a master mix of RT-LAMP and DETECTR assay reagents, including Cas12 variant (SEQ ID NO: 17) protein are reconstituted after lyophilization. The results of a reconstituted lyophilized Cas12 variant DETECTR master mix using SEQ ID NO: 17 are shown in
In some embodiments, the master mix of reagents and target for one assay is lyophilized. In some embodiments, the master mixes from more than one assay are pooled and lyophilized. In some embodiments, a duration of time occurs between mixing and lyophilization.
In some embodiments, lyophilized master mixes of reagents from more than one assay are prepared in volumes of less than 1 mL. In some embodiments, lyophilized master mixes of reagents from more than one assay are prepared in volumes of less than 250 uL. In some embodiments, lyophilized master mixes of reagents from more than one assay are prepared in volumes of less than 25 uL. In some embodiments, lyophilized master mixes of reagents from more than one assay are prepared in volumes of less than 10 uL.
In some embodiments, lyophilized master mixes of assay reagents are analyzed by dynamic scanning calorimetry (DSC).
In some embodiments, an excipient is used to stabilize the sample throughout the lyophilization process that may comprise freezing and drying steps.
In some embodiments, the hygroscopicity of enzymes and reagents is optimized to improve lyophilization performance.
LAMP Amplification with Cas14a DETECTR in Single Reaction Volume (One-Pot)
Described herein are various methods of sample amplification and detection in a single reaction volume. Any of the devices described herein may be configured to perform amplification and detection in a same well, chamber, channel, or volume in the device. In certain instances, methods include simultaneous amplification and detection in the same volume. In certain instances, methods include sequential amplification and detection in the same volume. In some embodiments, sample amplification may comprise LAMP amplification.
For some embodiments, RT-LAMP can be performed at lower temperatures by using Klenow (exo-) or Bsu polymerases: LowLAMP. RT-LAMP is normally performed at temperatures between 55C-70 C. Results can be seen in
The Cas14a1 sequence is:
In some embodiments, a tracrRNA, known as R1518 is used and is native for the system and has the sequence of:
In some embodiments, Cas14a. 1 uses two RNA components, a tracrRNA and a crRNA. In some embodiments, the native crRNA repeat that occurs with this system is used.
In some embodiments, crRNAs used are:
For some embodiments, buffers that were compatible with Cas14a and low temperature RT-LAMP (LowLAMP) were identified. Results are shown in
In some embodiment, primers and dNTPs have the greatest inhibitory effect on Cas 14 performance as seen in the results presented in
In some embodiments, LAMP functions with lower concentrations of dNTPs and primers as shown in
In some embodiments, One-pot Cas14 with LowLAMP was tested at 50 C. For such embodiments, one-pot DETECTR using Cas14 and LowLAMP at 50 C using a Klenow (exo-) DNA polymerase was shown to be functional as seen in
In some embodiments, Cas14 is used with a polymerase (e.g., a Bsm DNA polymerase, a Bst DNA polymerase, a Klenow (exo-) DNA polymerase, or a Bsu DNA polymerase) (55C) for the OnePot assay. The one-pot reaction may be faster if the reaction temperature is increased from 50 C to 55 C. However, the DNA polymerase used in LowLAMP is not functional at 55 C, so here Bsm DNA polymerase was used which works more robustly at 55 C than other LAMP polymerases such as Bst. Several different concentrations of dNTPs and primers were tested and performance of the assay was assessed. Results shown in
For some embodiments, the initial performance of the one-pot DETECTR reaction, called HotPot was evaluated. Results are shown in
For some embodiments, the limit of detection experiments were performed two different DNA polymerases at 55 C. Results are shown in
For various embodiments described herein, the assay turn-around time is 5 minutes. In some embodiments, the assay turn-around time is 10 minutes. In some embodiments, the turn-around time is 15 minutes. In some embodiments, the turn-around time is 20 minutes. In some embodiments, the turn-around time is 30 minutes. In some embodiments, the turn-around time is 40 minutes or less.
Described herein are various methods of sample amplification and detection in a single reaction volume. Any of the devices described herein may be configured to perform amplification and detection in a same well, chamber, channel, or volume in the device. In certain instances, methods include simultaneous amplification and detection in the same volume. In certain instances, methods include sequential amplification and detection in the same volume. In some embodiments, sample amplification may comprise NEAR amplification.
For some embodiments, replacing Bst polymerase in NEAR can enable SARS-CoV-2 detection at lower temperatures as shown in
For some embodiments, NEAR amplification functions in Cas14a optimal buffers as shown in
In some embodiments, Cas14a functions in a range of KOAc salt concentrations as shown in
In some embodiments, increasing concentrations of KOAc improve NEAR performance in Cas14a optimal buffers, as seen in
Performance of Cas14a.1 crRNAs on SARS-CoV-2 E-gene amplicon are shown in
In some embodiments, the performance of Klenow (exo-) NEAR assay in IB13 buffer at decreasing salt concentrations was evaluated as shown in
One-Pot sRCA: Rolling Circle Amplification with Cas14a DETECTR in Single Reaction Volume
Described herein are various methods of sample amplification and detection in a single reaction volume. Any of the devices described herein may be configured to perform amplification and detection in a same well, chamber, channel, or volume in the device. In certain instances, methods include simultaneous amplification and detection in the same volume. In certain instances, methods include sequential amplification and detection in the same volume. In some embodiments, sample amplification may comprise RCA.
In some embodiments, screening dumbbell DNA templates are screened for sRCA performance, as shown in
For some embodiments, the performance of Cas14a to detect product of RCA reaction was monitored, as seen in
In some embodiments, of the One-Pot assay sRCA, Cas14 is used. Functional results for such embodiments are shown in
In some embodiments, a trigger oligo is titrated for a Cas14 One-Pot sRCA assay. For this embodiment, the minimal concentration of trigger oligo that is required to initiate the one-pot Cas14 sRCA reaction was determined. Results shown in
In some embodiments, the Cas 12 variant enzyme (SEQ ID NO: 17) is used in the one-pot sRCA assay. In other embodiments, it has been shown that Cas14 is capable of functioning in a one-pot sRCA reaction. In this embodiment, it was shown that Cas12 variant (SEQ ID NO: 17) is also capable of functioning in this assay at 45 C. The results from the cleavage of ssDNA FQ reporter included in the sRCA reaction are shown in
In some embodiments, Cas13-compatible DNA template is used for RCA.
In some embodiments, the Cas 13M26 is used in the one-pot sRCA reaction.
CasPin: Cas13 Positive-Feedback Loop Leveraging Cas13 ssDNA Targeting
Described herein are various methods of signal amplification. Any of the devices described herein may be configured to perform signal amplification after the reporter has been cleaved by the programmable nuclease. Signal amplification may improve detection of rare targets in a complex sample. In certain instances, methods include leveraging ssDNA targeting of the programmable nuclease (e.g., Cas13) to create a positive feedback loop upon biding of the programmable nuclease to the target nucleic acid to cleave additional reporters and amplify the signal generated by the presence of the target nucleic acid.
In some embodiments, two hairpins are used on either end of the target site.
Described herein are various devices and methods for running one-pot DETECTR assays on a handheld device. Any of the devices described herein may be configured to perform a one-pot DETECTR assay. For example, the device shown in
Described herein are various methods of multiplexing detection. Any of the devices described herein may be configured for multiplexing (e.g., detecting multiple target nucleic acids). In certain instances, multiplexed detection may utilize one or more lateral flow assay strips.
Described here are various devices and methods for a DETECTR™ assay based multiplex lateral flow strip as illustrated in
In some embodiments, as illustrated in
In some embodiments, lateral flow assay strips (10710) are used to detect cleaved reporters (10709). In some embodiments, cleaved reporters (10709) are contacted to the sample pad (10711) of the lateral flow strip (10710). In some embodiments, the cleaved reporters (10709) bind to conjugate particles present in the sample pad. In some embodiments, the conjugate particles are gold nanoparticles. In some embodiments, the gold nanoparticles are functionalized with anti-biotin. In some embodiments, the anti-biotin functionalized gold nanoparticles bind to the cleaved reporter which contains one or more biotins in the binding moiety (10705).
In some embodiments, the reporter contains a second linker. In some embodiments, the second linker links one or more binding moieties to the nucleic acid. In some embodiments, the second linker links one or more labels to the nucleic acid. In some embodiments, the second linker links both one or more binding moieties and one or more labels to the nucleic acid of the reporter. In some embodiments, the reporter is a dendrimer or trebler molecule.
In some embodiments, the reporter contains a label. In some embodiments, label is FITC, DIG, TAMRA, Cy5, AF594, Cy3, or any appropriate label for a lateral flow assay.
In some embodiments, the reporter may comprise chemical functional group for binding. In some embodiments, the chemical functional group is biotin. In some embodiments, the chemical functional group is complimentary to a capture probe on the flowing capture probe (e.g., conjugate particle or capture molecule). In some embodiments, the flowing capture probe is a gold nanoparticle functionalized with anti-biotin. In some embodiments, the flow capture probe is located in the sample pad. In some embodiments, the flowing capture probe is located in a conjugate pad in contact with the sample pad, wherein both lateral flow assay strip may comprise both the sample pad and conjugate pad, further wherein both the sample pad and the conjugate pad are in fluid communication with the detection region.
In some embodiments, the lateral flow assay strip (10710) contains a detection region (10712). In some embodiments, the detection region (10712) may comprise one or more detection spots. In some embodiments, the detection spots contain a stationary capture probe (e.g., capture molecule). In some embodiments, the stationary capture probe may comprise one or more capture antibodies. In some embodiments, the capture antibodies are anti-FITC, anti-DIG, anti-TAMRA, anti-Cy5, anti-AF594, or any other appropriate capture antibody capable of binding the detection moiety or conjugate.
In some embodiments, the flowing capture probe comprising FITC is captured by a stationary capture probe comprising anti-FITC antibody. In some embodiments, the flowing capture probe comprising TAMRA is captured by a stationary capture probe comprising anti-TAMRA antibody. In some embodiments, the flowing capture probe comprising DIG is captured by the stationary capture probe comprising anti-DIG antibody. In some embodiments, the flowing capture probe comprising Cy5 is captured by the stationary capture probe comprising anti-Cy5 antibody. In some embodiments, the flowing capture probe comprising AF574 is captured by the stationary capture probe comprising anti-AF594 antibody.
In some embodiments, the lateral flow assay strip (10710) may comprise a control line (10714). In some embodiments, the control line (10714) may comprise anti-IgG that is complimentary to all flowing capture probes. In some embodiments, when a flowing capture probe does not bind to a reporter the flowing capture probe will be captured by the anti-IgG on the control line, ensuring the user that the device is working properly even no signal is read from the test line.
In some embodiments, the lateral flow assay strip (10710) may comprise a sample pad. In some embodiments, the flowing capture probe may comprise anti-biotin. In some embodiments, the flowing capture probe may comprise HRP. In some embodiments, the flowing capture probe may comprise HRP-anti-biotin. In some embodiments, the flowing capture probe is HRP-anti-biotin DAB/TMB.
Described here are various devices and methods for a DETECTR™ assay based multiplex lateral flow strip as illustrated in
Described herein are various embodiments of lateral flow-based detection as illustrated in
Described here are various methods and devices utilizing HRP-enhanced multiplexed DETECTR™ assays utilizing lateral flow assay strips for readout. In some embodiments, an HRP-signal enhanced multiplexed lateral flow assay as illustrated in
Described herein are various embodiments for multiplexed target nucleic acid detection utilizing Cas13 RNA cleaving specificity over DNA, HRP-signal enhancement, and capture oligo probe specificity. In some embodiments, as shown in
In some embodiments, one or more programmable nuclease probes (11300-11302) are used for guide pooling to achieve enhanced signal detection in lateral flow assays as shown in
Described herein are various embodiments of guide pooling to achieve enhanced signal detection in lateral flow assays. For some embodiments as described herein, guide pooling shows enhanced Cas12a activity.
Described herein are various methods and devices for a programmable nuclease (e.g., DETECTR) assay based multiplex lateral flow assay as illustrated in
In some embodiments, the amplification region (12004) may be coated. In some embodiments, a coating may be a hydrophilic coating, a hydrophobic coating, an inorganic coating, or an organic coating. In some embodiments, a coating may comprise a polymer coating. In some embodiments, a coating may comprise a polyethylene glycol coating. In some embodiments, a coating may comprise a streptavidin coating. In some embodiments, the interspersed polymers may be a crowding agent. In some embodiments, the crowding agent may comprise polyethylene glycol.
In some embodiments, the one or more reaction or heating zones may comprise guide nucleic acids (e.g, sgRNAs) immobilized to a surface (e.g., a glass bead disposed within a DETECTR zone). In some embodiments, the guide nucleic acids are part of a programmable nuclease (e.g., Cas-complex) probe immobilized to a surface. In some embodiments, a guide nucleic acid is designed to specifically bind to a target nucleic acid in the sample. In some embodiments, there are different guide nucleic acids corresponding to different locations on the surface and/or different surfaces in the one or more zones, where each different guide nucleic acid is complimentary for a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing guide nucleic acids, each surface location is functionalized with one or more reporters having distinct functional groups. In some embodiments, the reporters may be in close enough proximity to be cleaved by the programmable nuclease probes. In some embodiments, as described in example 13, reporters are cleaved and released into the solution upon binding between a particular guide nucleic acid and the target nucleic acid to which the guide nucleic acid is designed to specifically bind. In some embodiments, reporters are functionalized with a detection moiety (e.g., a label).
In some embodiments, chemical heating may be used. In some embodiments, chemical heating may be used to supply energy to initiate and run reactions. In some embodiments, chemical heating may be used to supply energy to initiate and run programmable nuclease assay reactions. In some embodiments, chemical heating may be used to heat reaction or heating zones. In some embodiments, chemical heating may be used to heat regions, chambers, volumes, zones, surfaces, or areas of a device.
In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region disposed downstream of the amplification region. Each lateral flow assay strip contains one or more detection regions or spots, where each detection region or spot contains a different type of capture antibody. In some embodiments, each lateral flow assay strip contains a different type of capture antibody. In some embodiments, each capture antibody type specifically binds to a particular label type of a reporter. In some embodiments, a first lateral flow assay strip contains the capture antibody anti-FITC. In some embodiments, a first DETECTR region or surface location (e.g., within a reaction chamber or heating region) contains the immobilized programmable nuclease (e.g., Cas-complex) including the guide nucleic acid (e.g., sgRNA) specific to the first target nucleic acid sequence. In some embodiments, the first DETECTR region or surface location additionally contains a first immobilized reporter which is labeled with a first detection moiety (e.g., FITC). In some embodiments, upon binding of the first target nucleic acid sequence, the first immobilized reporter is cleaved and released into solution. In some embodiments, the first detection moiety is released into solution and the remainder of the first reporter remains immobilized on the surface. Alternatively, or in combination, in some embodiments, a second lateral flow assay strip contains the capture antibody anti-DIG. In some embodiments, a second DETECTR region or surface location (e.g., within a reaction chamber or heating region) contains the immobilized programmable nuclease (e.g., Cas complex) including the guide nucleic acid (e.g., sgRNA) specific to the second target nucleic acid sequence. In some embodiments, the second DETECTR region or surface location additionally contains a second immobilized reporter which is labeled with a second detection moiety (e.g., DIG). Therefore, in some embodiments, upon binding of the second target nucleic acid sequence, the second immobilized reporter is cleaved and released into solution. In some embodiments, the second detection moiety is released into solution and the remainder of the second reporter remains immobilized on the surface.
In some embodiments, the solution containing the first and second cleaved reporters is transferred from the amplification region to the lateral flow region comprising the first lateral flow assay strip and the second lateral flow assay strip. In some embodiments, a chase buffer or diluent is introduced into a diluent input and negative pressure is applied to the negative pressure port to contact the solution containing the first and second cleaved reporters to the lateral flow assay strips of the lateral flow region, where the reporters are bound to conjugate molecules e.g., anti-biotin-AuNPs. In some embodiments, the AuNP-reporter conjugates having the first reporter labeled with the first detection moiety (e.g., FITC) will selectively bind to a first detection region or spot containing the first capture antibody (e.g., anti-FITC) on the first lateral flow assay strip, thus indicating the presence of the first target nucleic acid sequence in the sample. In some embodiments, the AuNP-reporter conjugates having mostly the second reporter labeled with the second detection moiety (e.g., DIG) will selectively bind to a second detection region or spot containing the second capture antibody (e.g., anti-DIG) on the second lateral flow assay strip, thus indicating the presence of the second target nucleic acid sequence in the sample. In this manner, for some embodiments, parallel detection of two or more target nucleic acid sequences present in a multiplexed sample is enabled.
In some embodiments, the amplification region is configured to hold about 200 μL of liquid (e.g., sample solution and reagent(s)). In some embodiments, each lateral flow assay strip is configured to hold about 80 μL of liquid (e.g., sample solution and/or chase buffer). In some embodiments, the device may comprise more than one lateral flow assay strip. For example, the device may comprise two, three, four, five, six, seven, eight, nine, ten, or more lateral flow assay strips. In some embodiments, one or more lateral flow assay strips are configured to detect a control sequence instead of or in addition to a target sequence. For example, a device comprising six lateral flow assay strips may comprise five lateral flow assay strips configured to detect one or more target sequences (e.g., five different target sequences) and one lateral flow assay strip configured to detect a control sequence.
Described herein are various methods and devices for a programmable nuclease assay based multiplex lateral flow assay as illustrated in
In some embodiments, the one or more programmable nuclease reagent(s) comprise guide nucleic acids immobilized to a surface (e.g., a glass bead). In some embodiments, the guide nucleic acids are part of a programmable nuclease (e.g., Cas-complex) probe immobilized to a surface. In some embodiments, a guide nucleic acid may be designed to specifically bind to a target nucleic acid in the sample. In some embodiments, there are different guide nucleic acids corresponding to different locations on the surface and/or different surfaces in the one or more zones, where each different guide nucleic acid is complimentary for a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing guide nucleic acids, each surface location may be functionalized with one or more reporters having distinct functional groups. In some embodiments, the reporters are in close enough proximity to be cleaved by the programmable nuclease probes. In some embodiments, as described in example 13, reporters are cleaved and released into the solution upon binding between a particular sgRNA and the target nucleic acid to which the guide nucleic acid is designed to specifically bind. In some embodiments, reporters are functionalized with a detection moiety (e.g., a label).
In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region disposed downstream of the amplification region. Each lateral flow assay strip contains one or more detection regions or spots, where each detection region or spot contains a different type of capture antibody. In some embodiments, each lateral flow assay strip may contain a different type of capture antibody. In some embodiments, each capture antibody type specifically binds to a particular label type of a reporter. In some embodiments, a first lateral flow assay strip contains a first capture antibody (e.g., anti-FITC). In some embodiments, a first surface (e.g., bead) contains the immobilized programmable nuclease (e.g., Cas-complex) including the guide nucleic acid (e.g., sgRNA) specific to the first target nucleic acid sequence. In some embodiments, the first surface additionally contains a first immobilized reporter which is labeled with a first detection moiety (e.g., FITC). In some embodiments, upon binding of the first target nucleic acid sequence, the first immobilized reporter is cleaved and released into solution as described herein. Alternatively, or in combination, in some embodiments, a second lateral flow assay strip contains a second capture antibody (e.g., anti-DIG). In some embodiments, a second surface (e.g., bead) contains the second immobilized programmable nuclease including the guide nucleic acid specific to the second target nucleic acid sequence. In some embodiments, the second surface additionally contains a second immobilized reporter which is labeled with a second detection moiety (e.g., DIG). Therefore, in some embodiments, upon binding of the second target nucleic acid sequence, the second immobilized reporter is cleaved and released into solution.
In some embodiments, the solution containing the first and second cleaved reporters is transferred from the amplification region to the lateral flow region comprising the first lateral flow assay strip and the second lateral flow assay strip. In some embodiments, a chase buffer or diluent is introduced into a diluent input or reservoir and negative pressure is applied to the negative pressure port to contact lateral flow region. A pressure valve may be disposed between the amplification region and the lateral flow region in order to regulate flow of the sample solution from the amplification region to the lateral flow region before amplification has occurred. Actuation of the pressure valve enables the solution containing the first and second cleaved reporters to contact the lateral flow assay strips of the lateral flow region, where the reporters are bound to conjugate molecules, e.g., anti-biotin-AuNPs. In some embodiments, the AuNP-reporter conjugates having the first reporter labeled with the first detection moiety (e.g., FITC) will selectively bind to a first detection region or spot containing a first capture antibody (e.g., anti-FITC) on the first lateral flow assay strip, thus indicating the presence of the first target nucleic acid sequence in the sample. In some embodiments, the AuNP-reporter conjugates having mostly the second reporter labeled with the second detection moiety (e.g., DIG) will selectively bind to a second detection region or spot containing a second capture antibody (e.g., anti-DIG) on the second lateral flow assay strip, thus indicating the presence of the second target nucleic acid sequence in the sample. In this manner, for some embodiments, parallel detection of two or more target nucleic acid sequences present in a multiplexed sample is enabled.
In some embodiments, the amplification region is configured to hold about 200 μL of liquid (e.g., sample solution and reagent(s)). In some embodiments, each lateral flow assay strip is configured to hold about 80 μL of liquid (e.g., sample solution and/or chase buffer). In some embodiments, the device may comprise more than one lateral flow assay strip. For example, the device may comprise two, three, four, five, six, seven, eight, nine, ten, or more lateral flow assay strips. In some embodiments, one or more lateral flow assay strips are configured to detect a control sequence instead of or in addition to a target sequence. For example, a device comprising six lateral flow assay strips may comprise five lateral flow assay strips configured to detect one or more target sequences (e.g., five different target sequences) and one lateral flow assay strip configured to detect a control sequence.
Described herein are various methods and devices for a DETECTR assay based multiplex lateral flow assay as illustrated in
Described herein are various methods and devices for a handheld assay device as illustrated in
Described herein are various methods and devices for a handheld assay device as illustrated in
Described herein are various methods and devices for a handheld assay device as illustrated in
Disclosed herein are methods for optimizing proteins and formulations for diagnostic applications. An ideal Cas protein for CRISPR diagnostics is one that is fast, robust, and sensitive. A fast protein enables reduced turnaround times for diagnostic assays. Robust enzymes are more likely to be successful when combined with other molecular processes, such as in the one-pot DETECTR assay. Sensitive enzymes enable lower limits of detection from either small amounts of amplified product or when eliminating pre-amplification and doing direct detection of target nucleic acids.
Initial optimization of programmable nuclease systems (e.g., the combination of Cas protein and guide nucleic acids) involved screening the performance of candidate systems in a variety of buffers and at temperatures from 35° C. to 60° C. The buffers selected for screening included buffers used in DETECTR assays and other Cas activity buffers. The buffers may comprise a range of salt types, salt concentrations, counter ion types, and various additives important for polymerase performance.
In some embodiments, the device includes multiple lateral flow strips. Each reaction chamber may be configured to interface with a detection region comprising a lateral flow strip. In some embodiments, the detection region may be in fluid communication with the reaction chamber. In some embodiments, the detection region may be contacted to the reaction chamber after amplification and the programmable nuclease-based reactions have been performed in the reaction chamber. In some embodiments, the lateral flow strips may be configured to be inserted into the reaction chambers as shown in
In some embodiments, each reaction chamber may comprise one or more guide nucleic acids (e.g., sgRNAs) immobilized to a surface (e.g., a glass bead or hydrogel disposed within a reaction chamber). In some embodiments, the guide nucleic acids are part of a programmable nuclease (e.g., Cas-complex) probe immobilized to a surface. In some embodiments, a guide nucleic acid is designed to specifically bind to a target nucleic acid in the sample. In some embodiments, there are different guide nucleic acids corresponding to different reaction chambers, where each different guide nucleic acid is complimentary for a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing guide nucleic acids, each reaction chamber may contain or be functionalized with one or more reporters having distinct functional groups as described herein. In some embodiments, the reporters may be in close enough proximity to be cleaved by the programmable nuclease probes. In some embodiments, as described in example 13, reporters are cleaved and portion thereof (e.g., a detection moiety) is released into the solution upon binding between a particular guide nucleic acid and the target nucleic acid to which the guide nucleic acid is designed to specifically bind. In some embodiments, reporters are functionalized with a detection moiety (e.g., a label).
In some embodiments, chemical heating may be used. In some embodiments, chemical heating may be used to supply energy to initiate and run reactions. In some embodiments, chemical heating may be used to supply energy to initiate and run programmable nuclease assay reactions. In some embodiments, chemical heating may be used to heat reaction or heating zones. In some embodiments, chemical heating may be used to heat regions, chambers, volumes, zones, surfaces, or areas of a device.
In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region disposed downstream of the reaction chamber. In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region which may be brought into fluid communication with the reaction chamber. Each lateral flow assay strip contains one or more detection regions or spots, where each detection region or spot contains a different type of capture antibody. In some embodiments, each lateral flow assay strip contains a different type of capture antibody. In some embodiments, each capture antibody type specifically binds to a particular label type of a reporter. In some embodiments, a first lateral flow assay strip contains the capture antibody anti-FITC. In some embodiments, a first DETECTR region or surface location (e.g., within a first reaction chamber) contains the immobilized programmable nuclease (e.g., Cas-complex) including the guide nucleic acid (e.g., sgRNA) specific to the first target nucleic acid sequence. In some embodiments, the first DETECTR region or surface location additionally contains a first immobilized reporter which is labeled with a first detection moiety (e.g., FITC). In some embodiments, upon binding of the first target nucleic acid sequence, the first immobilized reporter is cleaved and released into solution. In some embodiments, the first detection moiety is released into solution and the remainder of the first reporter remains immobilized on the surface. Alternatively, or in combination, in some embodiments, a second lateral flow assay strip contains the capture antibody anti-DIG. In some embodiments, a second DETECTR region or surface location (e.g., within a second reaction chamber) contains the immobilized programmable nuclease (e.g., Cas complex) including the guide nucleic acid (e.g., sgRNA) specific to the second target nucleic acid sequence. In some embodiments, the second DETECTR region or surface location additionally contains a second immobilized reporter which is labeled with a second detection moiety (e.g., DIG). Therefore, in some embodiments, upon binding of the second target nucleic acid sequence, the second immobilized reporter is cleaved and released into solution. In some embodiments, the second detection moiety is released into solution and the remainder of the second reporter remains immobilized on the surface.
In some embodiments, the solutions containing the first or second cleaved reporters are transferred from their respective reaction chambers to a first lateral flow assay strip and a second lateral flow assay strip, respectively. In some embodiments, a chase buffer or diluent is introduced into a diluent input and negative pressure is applied to the negative pressure port to contact the solutions containing the first or second cleaved reporters to their respective lateral flow assay strips. The reporters may be bound to conjugate molecules e.g., anti-biotin-AuNPs. In some embodiments, the AuNP-reporter conjugates having the first reporter labeled with the first detection moiety (e.g., FITC) will selectively bind to a first detection region or spot containing the first capture antibody (e.g., anti-FITC) on the first lateral flow assay strip, thus indicating the presence of the first target nucleic acid sequence in the sample. In some embodiments, the AuNP-reporter conjugates having mostly the second reporter labeled with the second detection moiety (e.g., DIG) will selectively bind to a second detection region or spot containing the second capture antibody (e.g., anti-DIG) on the second lateral flow assay strip, thus indicating the presence of the second target nucleic acid sequence in the sample. In this manner, for some embodiments, parallel detection of two or more target nucleic acid sequences present in a multiplexed sample is enabled.
In some embodiments, the device may comprise more than one lateral flow assay strip. For example, the device may comprise two, three, four, five, six, seven, eight, nine, ten, or more lateral flow assay strips. In some embodiments, each reaction chamber may be interfaced with a lateral flow assay strip for multiplexing. In some embodiments, one or more lateral flow assay strips are configured to detect a control sequence instead of or in addition to a target sequence. For example, a device comprising six lateral flow assay strips may comprise five lateral flow assay strips configured to detect one or more target sequences (e.g., five different target sequences) and one lateral flow assay strip configured to detect a control sequence.
Described herein are various methods and devices for chemical heating-based PON devices utilizing lateral flow assay strips for DETECTR readout.
In some embodiments, a sample contains one or more target nucleic acid sequences. In some embodiments, the sample is introduced into a sample input (also referred to herein as a sample interface) and loaded into a plurality of sample loading chambers via a sample loading channel. In some embodiments, the sample interface may be configured to concentrate, filter, lyse, or otherwise prepare the sample as described herein. In some embodiments, the sample may be loaded into the sample loading channel and sample loading chambers automatically via gravity and/or capillary forces. Alternatively, or in combination, positive pressure may be applied to the sample interface by a positive pressure source (e.g., a syringe) in order to load the sample loading channel and sample loading chambers with the sample. In some embodiments, the sample is a lysis sample. The sample loading channel may be configured (e.g., sized and shaped) to load the plurality of sample loading chambers with a pre-determined volume of sample liquid. Upon loading of the sample loading chambers, sufficient positive pressure may be applied to the sample loading channel and chambers in order to break the pressure cracking valves coupled to each sample loading chamber. Sample liquid is then transferred downstream of each sample loading chamber to a reaction chamber via a fluid channel. In some embodiments, a downstream end of the fluid channel may comprise a valve, hydrophobic filter, or frit or the like configured to allow gas to pass through during positive pressure application to the sample interface and enable fluid to flow along the fluid channel unimpeded. The hydrophobic filter or frit may be configured to prevent liquid to pass through as the sample is moved into the reaction chamber. In some embodiments, the sample input, sample loading channel(s), and/or sample loading chamber(s) may be located in a sample loading layer of the device. In some embodiments, the sample loading layer may be fluidly coupled to the reaction chambers. In some embodiments, the sample loading layer may be disposed above the reaction chambers. In some embodiments, the sample loading layer may be a chip or microfluidic device assembled over a housing comprising the reaction chambers. In some embodiments, each reaction chamber is fluidly independent of every other reaction chamber in order to facilitate target analysis multiplexing. The reaction chambers may comprise one or more dried or lyophilized programmable nuclease reagents (e.g., immobilized to a glass bead) and/or one or more liquid, dried, or lyophilized amplification reagents (e.g., powdered). In some embodiments, the programmable nuclease reagents and/or amplification reagents may be added by a user prior to use (e.g., pipetted into the reaction chambers by a user prior to connecting a sample loading layer thereto) and/or added during manufacturing. In some embodiments, amplification of one or more target nucleic acids is performed in each reaction chamber. In some embodiments, the amplification reagents of each reaction chamber may be different and may be designed to amplify different target nucleic acids of a plurality of target nucleic acids. The reaction chambers may be heated (e.g., to 55 C for 20 minutes or less) to amplify the one or more target nucleic acids as described herein. In some embodiments, heating may facilitate programmable nuclease-based detection reaction as described herein. Heating can include liquid-phase chemical heating, solid phase chemical heating, and electric heating (including, but not limited to, resistance/Joule heating, induction heating, Peltier heat pumping, etc.). In some embodiments, the reaction chambers may be surrounded by or disposed in a heating reservoir or zone. For example, the reaction chambers may comprise vertical chambers which are substantially surrounded by a heating reservoir as shown in
In some embodiments, the device includes multiple lateral flow strips. Each reaction chamber may be configured to interface with a detection region comprising a lateral flow strip. In some embodiments, the detection region may be in fluid communication with the reaction chamber. In some embodiments, the detection region may be contacted to the reaction chamber after amplification and the programmable nuclease-based reactions have been performed in the reaction chamber. In some embodiments, the lateral flow strips may be configured to be inserted into the reaction chambers as shown in
In some embodiments, each reaction chamber may comprise one or more guide nucleic acids (e.g., sgRNAs) immobilized to a surface (e.g., a glass bead or hydrogel disposed within a reaction chamber). In some embodiments, the guide nucleic acids are part of a programmable nuclease (e.g., Cas-complex) probe immobilized to a surface. In some embodiments, a guide nucleic acid is designed to specifically bind to a target nucleic acid in the sample. In some embodiments, there are different guide nucleic acids corresponding to different reaction chambers, where each different guide nucleic acid is complimentary for a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing guide nucleic acids, each reaction chamber may contain or be functionalized with one or more reporters having distinct functional groups as described herein. In some embodiments, the reporters may be in close enough proximity to be cleaved by the programmable nuclease probes. In some embodiments, as described in example 13, reporters are cleaved and portion thereof (e.g., a detection moiety) is released into the solution upon binding between a particular guide nucleic acid and the target nucleic acid to which the guide nucleic acid is designed to specifically bind. In some embodiments, reporters are functionalized with a detection moiety (e.g., a label).
In some embodiments, chemical heating may be used. In some embodiments, chemical heating may be used to supply energy to initiate and run reactions. In some embodiments, chemical heating may be used to supply energy to initiate and run programmable nuclease assay reactions. In some embodiments, chemical heating may be used to heat reaction or heating zones. In some embodiments, chemical heating may be used to heat regions, chambers, volumes, zones, surfaces, or areas of a device. In some embodiments, the reaction chambers may be surrounded by or disposed in a heating reservoir or zone. The heating reservoir may comprise a liquid-phase chemical heating agent configured to generate an exothermic chemical reaction upon contact with a nucleation site. For example, a piercer may be configured to pierce through a film surrounding the heating reservoir and contact the liquid-phase chemical heating agent (e.g., sodium acetate). The piercer may provide a nucleation site for crystallization of the liquid-phase chemical heating agent to begin phase change transformation and thereby generate heat within the heating reservoir. The amount of chemical heating agent may be adjusted to provide a desired temperature and/or duration of heating to the reaction chambers.
In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region disposed downstream of the reaction chamber. In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region which may be brought into fluid communication with the reaction chamber. In some embodiments, the detection region comprises a detection tray. Each lateral flow assay strip contains one or more detection regions or spots, where each detection region or spot contains a different type of capture antibody. In some embodiments, each lateral flow assay strip contains a different type of capture antibody. In some embodiments, each capture antibody type specifically binds to a particular label type of a reporter. In some embodiments, a first lateral flow assay strip contains the capture antibody anti-FITC. In some embodiments, a first DETECTR region or surface location (e.g., within a first reaction chamber) contains the immobilized programmable nuclease (e.g., Cas-complex) including the guide nucleic acid (e.g., sgRNA) specific to the first target nucleic acid sequence. In some embodiments, the first DETECTR region or surface location additionally contains a first immobilized reporter which is labeled with a first detection moiety (e.g., FITC). In some embodiments, the first reporter may be immobilized within a hydrogel as described herein. In some embodiments, upon binding of the first target nucleic acid sequence, the first immobilized reporter is cleaved and released into solution. In some embodiments, the first detection moiety is released into solution and the remainder of the first reporter remains immobilized on the surface. Alternatively, or in combination, in some embodiments, a second lateral flow assay strip contains the capture antibody anti-DIG. In some embodiments, a second DETECTR region or surface location (e.g., within a second reaction chamber) contains the immobilized programmable nuclease (e.g., Cas complex) including the guide nucleic acid (e.g., sgRNA) specific to the second target nucleic acid sequence. In some embodiments, the second DETECTR region or surface location additionally contains a second immobilized reporter which is labeled with a second detection moiety (e.g., DIG). In some embodiments, the second reporter may be immobilized within a hydrogel as described herein. Therefore, in some embodiments, upon binding of the second target nucleic acid sequence, the second immobilized reporter is cleaved and released into solution. In some embodiments, the second detection moiety is released into solution and the remainder of the second reporter remains immobilized on the surface.
In some embodiments, the solutions containing the first or second cleaved reporters are transferred from their respective reaction chambers to a first lateral flow assay strip and a second lateral flow assay strip, respectively. In some embodiments, a chase buffer or diluent is introduced into a diluent input and pressure is applied to contact the solutions containing the first or second cleaved reporters to their respective lateral flow assay strips. In some embodiments, the reaction chambers are vertical reaction chambers and gravity is used to provide fluid flow between the reaction chambers and their respective lateral flow assay strips. The reporters may be bound to conjugate molecules e.g., anti-biotin-AuNPs. In some embodiments, the AuNP-reporter conjugates having the first reporter labeled with the first detection moiety (e.g., FITC) will selectively bind to a first detection region or spot containing the first capture antibody (e.g., anti-FITC) on the first lateral flow assay strip, thus indicating the presence of the first target nucleic acid sequence in the sample. In some embodiments, the AuNP-reporter conjugates having mostly the second reporter labeled with the second detection moiety (e.g., DIG) will selectively bind to a second detection region or spot containing the second capture antibody (e.g., anti-DIG) on the second lateral flow assay strip, thus indicating the presence of the second target nucleic acid sequence in the sample. In this manner, for some embodiments, parallel detection of two or more target nucleic acid sequences present in a multiplexed sample is enabled.
In some embodiments, the device may comprise more than one lateral flow assay strip. For example, the device may comprise two, three, four, five, six, seven, eight, nine, ten, or more lateral flow assay strips. In some embodiments, each reaction chamber may be interfaced with a lateral flow assay strip for multiplexing. In some embodiments, one or more lateral flow assay strips are configured to detect a control sequence instead of or in addition to a target sequence. For example, a device comprising six lateral flow assay strips may comprise five lateral flow assay strips configured to detect one or more target sequences (e.g., five different target sequences) and one lateral flow assay strip configured to detect a control sequence.
In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in about 40 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in less than about 40 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in greater than about 40 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in about 25 minutes to about 35 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in about 35 minutes to about 45 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in about 45 minutes to about 55 minutes.
In some embodiments, results may be ready in less than about 1 minute after the sample begins the capillary process across the lateral flow assay strip. In some embodiments, results may be ready in less than about 2 minutes after the sample begins the capillary process across the lateral flow assay strip. In some embodiments, results may be ready in less than about 3 minutes after the sample begins the capillary process across the lateral flow assay strip. In some embodiments, results may be ready in less than about 5 minutes after the sample begins the capillary process across the lateral flow assay strip.
Described herein are various methods and devices for chemical heating-based PON devices utilizing lateral flow assay strips for DETECTR readout.
In some embodiments, a sample contains one or more target nucleic acid sequences. In some embodiments, the sample is introduced into a sample input (also referred to herein as a sample interface) and loaded into a plurality of sample loading chambers via a sample loading channel. In some embodiments, the sample interface may be configured to concentrate, filter, lyse, or otherwise prepare the sample as described herein. In some embodiments, a separate sample preparation reservoir may be provided and configured to concentrate, filter, lyse, or otherwise prepare the sample as described herein before the sample is loaded into the sample interface. In some embodiments, the sample may be loaded into the sample loading channel and sample loading chambers automatically via gravity and/or capillary forces. Alternatively, or in combination, negative pressure may be applied to the detection interface by a negative pressure source (e.g., a syringe) in order to load the sample loading channel and sample loading chambers with the sample. In some embodiments, the negative pressure source may be in the form of a detection pen comprising a lateral flow assay strip disposed therein. Counter-clockwise, upwards rotation of the actuator cap of the detection pen from a first configuration to a second configuration may draw fluid into the reaction channel or chamber from the sample interface. In some embodiments, the sample is a lysis sample. In some embodiments, the device may comprise a single reaction channel or chamber. In some embodiments, the device may comprise a plurality of reaction channels or chambers. In some embodiments, each reaction chamber is fluidly independent of every other reaction chamber in order to facilitate target analysis multiplexing. The reaction chamber(s) may comprise one or more dried or lyophilized programmable nuclease reagents (e.g., immobilized to a glass bead) and/or one or more liquid, dried, or lyophilized amplification reagents (e.g., powdered). In some embodiments, the programmable nuclease reagents and/or amplification reagents may be added by a user prior to use and/or added during manufacturing. In some embodiments, amplification of one or more target nucleic acids is performed in each reaction chamber. In some embodiments, the amplification reagents of each reaction chamber may be different and may be designed to amplify different target nucleic acids of a plurality of target nucleic acids. The reaction chambers may be heated (e.g., to 55 C for 20 minutes or less) to amplify the one or more target nucleic acids as described herein. In some embodiments, heating may facilitate programmable nuclease-based detection reaction as described herein. Heating can include liquid-phase chemical heating, solid phase chemical heating, and electric heating (including, but not limited to, resistance/Joule heating, induction heating, Peltier heat pumping, etc.). In some embodiments, the reaction chambers may be surrounded by or disposed in a heating reservoir or zone. For example, the reaction chambers may comprise serpentine channels or spiral tubes which are substantially surrounded by a heating reservoir as shown in
In some embodiments, the device includes one or more lateral flow strips disposed within one or more detection pens. Each reaction chamber may be configured to interface with a detection region comprising a lateral flow strip. In some embodiments, the detection region may be in fluid communication with the reaction chamber. In some embodiments, the detection region may be contacted to the reaction chamber after amplification and the programmable nuclease-based reactions have been performed in the reaction chamber. In some embodiments, the lateral flow strip may be configured to be inserted adjacent or into the reaction chamber/channel as shown in
In some embodiments, each reaction chamber may comprise one or more guide nucleic acids (e.g., sgRNAs) immobilized to a surface (e.g., a glass bead or hydrogel disposed within a reaction chamber). In some embodiments, the guide nucleic acids are part of a programmable nuclease (e.g., Cas-complex) probe immobilized to a surface. In some embodiments, a guide nucleic acid is designed to specifically bind to a target nucleic acid in the sample. In some embodiments, there are different guide nucleic acids corresponding to different reaction chambers, where each different guide nucleic acid is complimentary for a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing guide nucleic acids, each reaction chamber may contain or be functionalized with one or more reporters having distinct functional groups as described herein. In some embodiments, the reporters may be in close enough proximity to be cleaved by the programmable nuclease probes. In some embodiments, as described in example 13, reporters are cleaved and portion thereof (e.g., a detection moiety) is released into the solution upon binding between a particular guide nucleic acid and the target nucleic acid to which the guide nucleic acid is designed to specifically bind. In some embodiments, reporters are functionalized with a detection moiety (e.g., a label). In some embodiments, chemical heating may be used. In some embodiments, chemical heating may be used to supply energy to initiate and run reactions. In some embodiments, chemical heating may be used to supply energy to initiate and run programmable nuclease assay reactions. In some embodiments, chemical heating may be used to heat reaction or heating zones. In some embodiments, chemical heating may be used to heat regions, chambers, volumes, zones, surfaces, or areas of a device. In some embodiments, the reaction chambers may be surrounded by or disposed in a heating reservoir or zone. The heating reservoir may comprise a liquid-phase chemical heating agent configured to generate an exothermic chemical reaction upon contact with a nucleation site. For example, a piercer may be configured to pierce through a film surrounding the heating reservoir and contact the liquid-phase chemical heating agent (e.g., sodium acetate). The piercer may provide a nucleation site for crystallization of the liquid-phase chemical heating agent to begin phase change transformation and thereby generate heat within the heating reservoir. The amount of chemical heating agent may be adjusted to provide a desired temperature and/or duration of heating to the reaction chambers.
In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region disposed downstream of the reaction chamber. In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region which may be brought into fluid communication with the reaction chamber. In some embodiments, the detection region comprises a detection tray. Each lateral flow assay strip contains one or more detection regions or spots, where each detection region or spot contains a different type of capture antibody. In some embodiments, each lateral flow assay strip contains a different type of capture antibody. In some embodiments, each capture antibody type specifically binds to a particular label type of a reporter. In some embodiments, a first lateral flow assay strip contains the capture antibody anti-FITC. In some embodiments, a first DETECTR region or surface location (e.g., within a first reaction chamber) contains the immobilized programmable nuclease (e.g., Cas-complex) including the guide nucleic acid (e.g., sgRNA) specific to the first target nucleic acid sequence. In some embodiments, the first DETECTR region or surface location additionally contains a first immobilized reporter which is labeled with a first detection moiety (e.g., FITC). In some embodiments, the first reporter may be immobilized within a hydrogel as described herein. In some embodiments, upon binding of the first target nucleic acid sequence, the first immobilized reporter is cleaved and released into solution. In some embodiments, the first detection moiety is released into solution and the remainder of the first reporter remains immobilized on the surface. Alternatively, or in combination, in some embodiments, a second lateral flow assay strip contains the capture antibody anti-DIG. In some embodiments, a second DETECTR region or surface location (e.g., within a second reaction chamber) contains the immobilized programmable nuclease (e.g., Cas complex) including the guide nucleic acid (e.g., sgRNA) specific to the second target nucleic acid sequence. In some embodiments, the second DETECTR region or surface location additionally contains a second immobilized reporter which is labeled with a second detection moiety (e.g., DIG). In some embodiments, the second reporter may be immobilized within a hydrogel as described herein. Therefore, in some embodiments, upon binding of the second target nucleic acid sequence, the second immobilized reporter is cleaved and released into solution. In some embodiments, the second detection moiety is released into solution and the remainder of the second reporter remains immobilized on the surface.
In some embodiments, the solutions containing the first or second cleaved reporters are transferred from their respective reaction chambers to a first lateral flow assay strip and a second lateral flow assay strip, respectively. In some embodiments, a chase buffer or diluent is introduced into a diluent input and pressure is applied to contact the solutions containing the first or second cleaved reporters to their respective lateral flow assay strips. In some embodiments, the reaction chambers are vertical reaction chambers and gravity is used to provide fluid flow between the reaction chambers and their respective lateral flow assay strips. The reporters may be bound to conjugate molecules e.g., anti-biotin-AuNPs. In some embodiments, the AuNP-reporter conjugates having the first reporter labeled with the first detection moiety (e.g., FITC) will selectively bind to a first detection region or spot containing the first capture antibody (e.g., anti-FITC) on the first lateral flow assay strip, thus indicating the presence of the first target nucleic acid sequence in the sample. In some embodiments, the AuNP-reporter conjugates having mostly the second reporter labeled with the second detection moiety (e.g., DIG) will selectively bind to a second detection region or spot containing the second capture antibody (e.g., anti-DIG) on the second lateral flow assay strip, thus indicating the presence of the second target nucleic acid sequence in the sample. In this manner, for some embodiments, parallel detection of two or more target nucleic acid sequences present in a multiplexed sample is enabled.
In some embodiments, the device may comprise more than one lateral flow assay strip. For example, the device may comprise two, three, four, five, six, seven, eight, nine, ten, or more lateral flow assay strips. In some embodiments, each reaction chamber may be interfaced with a lateral flow assay strip for multiplexing. In some embodiments, one or more lateral flow assay strips are configured to detect a control sequence instead of or in addition to a target sequence. For example, a device comprising six lateral flow assay strips may comprise five lateral flow assay strips configured to detect one or more target sequences (e.g., five different target sequences) and one lateral flow assay strip configured to detect a control sequence.
In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in about 40 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in less than about 40 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in greater than about 40 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in about 25 minutes to about 35 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in about 35 minutes to about 45 minutes. In some embodiments, the isothermal amplification and/or programmable nuclease-based reaction may be completed in about 45 minutes to about 55 minutes.
In some embodiments, results may be ready in less than about 1 minute after the sample begins the capillary process across the lateral flow assay strip. In some embodiments, results may be ready in less than about 2 minutes after the sample begins the capillary process across the lateral flow assay strip. In some embodiments, results may be ready in less than about 3 minutes after the sample begins the capillary process across the lateral flow assay strip. In some embodiments, results may be ready in less than about 5 minutes after the sample begins the capillary process across the lateral flow assay strip.
Described herein are various methods and devices for chemical heating-based PON devices utilizing lateral flow assay strips for DETECTR readout.
Disclosed herein are systems, compositions, and methods for amplifying a signal from a target nucleic acid using an enzyme. In some embodiments, enzyme-based assays may allow for signal amplification of a binding event between one or more programmable nuclease probes and one or more target nucleic acids.
Disclosed herein are some strategies that use enzymes with programmable nucleases (e.g., DETECTR based assays) to amplify detection signals indicative of the presence of one or more target nucleic acids in a sample.
In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid, a programmable nuclease complex configured to bind to the target nucleic acid, and a reporter comprising an enzyme immobilized on a surface, and (b) adding an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.
In some embodiments, the method may comprise (a) providing a solution comprising a programmable nuclease complex configured to bind to a target nucleic acid and a reporter comprising an enzyme immobilized on a surface, and (b) adding the target nucleic acid and an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.
In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid and a reporter comprising an enzyme immobilized on a surface, and (b) adding a programmable nuclease complex configured to bind to the target nucleic acid and an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.
In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid and a programmable nuclease complex configured to bind to the target nucleic acid, and (b) adding a reporter comprising an enzyme immobilized on a surface and an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.
In some embodiments, the method may comprise (a) providing a solution comprising a reporter comprising an enzyme immobilized on a surface, and (b) adding a target nucleic acid, a programmable nuclease complex configured to bind to the target nucleic acid, and an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.
In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid, a programmable nuclease complex configured to bind to the target nucleic acid, and an enzyme substrate configured to be acted on by an enzyme to induce color change of the solution, and (b) adding reporter comprising the enzyme immobilized on one or more surfaces.
In some embodiments, the method may comprise (a) providing a solution comprising programmable nuclease complex configured to bind to a target nucleic acid and an enzyme substrate configured to be acted on by an enzyme to induce color change of the solution, and (b) adding the target nucleic acid and a reporter comprising the enzyme immobilized on one or more surfaces.
In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid and an enzyme substrate configured to be acted on by an enzyme to induce color change of the solution, and (b) adding a programmable nuclease complex configured to bind to the target nucleic acid and a reporter comprising the enzyme immobilized on one or more surfaces.
In some embodiments, the method may comprise (a) providing a solution comprising substrates configured to be acted on by an enzyme to induce color change of the solution, and (b) adding a target nucleic acid, a programmable nuclease complex configured to bind to the target nucleic acid, and a reporter comprising the enzyme immobilized on one or more surfaces.
In some embodiments, the reporter may comprise any of the reporters disclosed herein. In some embodiments, the programmable nuclease may comprise any one of the programmable nucleases disclosed herein. In some embodiments, the nucleic acid tether may comprise any of the nucleic acid tethers disclosed herein.
The nucleic acid tether of the reporter may be attached to, bound to, coupled to, or otherwise immobilized on the surface using any of the immobilization chemistries or linkages described herein. In some embodiments, the immobilization chemistry or linkage may be between: streptavidin and biotin; an amine group and N-hydroxysuccinimide (NHS), an amine group and an epoxy group, or a malemide group and a thiol group.
In some embodiments, the surface may be any surface disclosed herein. In some embodiments the surface may be of a support as described herein. In some embodiments, the support may comprise glass, PDMS, a hydrogel, plastic, magnetic beads, agarose beads, nitrocellulose, or any other support whose surface can be modified to bind nucleic acids. In some embodiments, the surface of the support, or of a reaction chamber, comprises streptavidin, biotin, an amine group, a carboxyl group, an epoxy group, an NHS group, a malemide group, or a thiol group. In some embodiments, the target nucleic acid may be any target nucleic acid disclosed herein. In some embodiments, the target nucleic acid may be a single-stranded DNA, a single-stranded RNA, a double-stranded DNA, a double-stranded RNA, or a double-stranded DNA/RNA hybrid.
In some embodiments, the guide nucleic acid may be any guide nucleic acid disclosed herein. In some embodiments, the guide nucleic acid may be a single-stranded DNA or a single-stranded RNA. In some embodiments, the nucleic acid tethering the reporter may comprise any of the reporter nucleic acids described herein.
In some embodiments, the enzyme may be HRP, AP, beta-galactosidase, acetylcholinesterase, catalase, catacolase, tyronase, nitrocefelin, alkaline phosphatase, or invertase. In some embodiments, the enzyme may be any one of the enzymes described herein.
In some embodiments, the enzyme may bind with an enzyme substrate and produce a detectable signal. In some embodiments, the enzyme substrate may be 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), p-Nitrophenyl Phosphate (PNPP), o-nitrophenyl-β-D-galactopyranoside (ONPG), 3,3′-diaminobenzidine (DAB), p-hydroxyphenylacetic acid, 3-(p-hydroxyphenyl)-propionic acid, homovanillic acid, or o-aminophenol. In some embodiments, the enzyme substrate may be a commercial enzyme substrate including SuperSignal ELISA Pico, SuperSignal Elisa Femto, CDP-Star Substrate, CSPD Substrate, DynaLight Substrate with RapidGlow Enhancer, QuantaBlu, QuantaRed, or Amplex.
In some embodiments, the enzyme may generate a colorimetric signal, a fluorescent signal, an electrochemical signal, a chemiluminescent signal, or another type of signal. In some embodiments, the enzyme may induce color-change in substances.
In some embodiments, the programmable nuclease of the programmable nuclease-enzyme fusion protein may be Cas12, Cas13, Cas14, or CasPhi. In some embodiments, the programmable nuclease of the programmable nuclease-enzyme fusion protein may be any programmable nuclease disclosed herein.
In some embodiments, the enzyme of the programmable nuclease-enzyme fusion protein may be HRP, AP, beta-galactosidase, acetylcholinesterase, or catalase. In some embodiments, the enzyme of the programmable nuclease-enzyme fusion protein may be any enzyme disclosed herein.
In some embodiments, the programmable nuclease may be fused by a peptide linker to the enzyme. In some embodiments, the C-terminus of the programmable nuclease portion may be fused by a peptide linker to the N-terminus of the enzyme. In some embodiments, the N-terminus of the programmable nuclease portion may be fused by a peptide linker to the C-terminus of the enzyme. In some embodiments, the peptide linker may be XTEN.
In some embodiments, the method may comprise (a) providing a solution comprising an enzyme substrate and a programmable nuclease-enzyme fusion protein immobilized on a surface, wherein the programmable nuclease-enzyme fusion protein comprises a programmable nuclease and an enzyme, wherein the programmable nuclease is configured to bind to a target nucleic acid, and (b) adding the target nucleic acid.
In some embodiments, the method may comprise (a) providing a solution comprising an enzyme substrate and a target nucleic acid, and (b) adding a programmable nuclease-enzyme fusion protein immobilized on a surface, wherein the programmable nuclease-enzyme fusion protein comprises a programmable nuclease and an enzyme, wherein the programmable nuclease is configured to bind to a target nucleic acid.
In some embodiments, the programmable nuclease-enzyme fusion protein may comprise any one of the programmable nucleases disclosed herein. In some embodiments, the target nucleic acid may be any one of the target nucleic acids disclosed herein. In some embodiments, the guide nucleic acid may comprise any one of the guide nucleic acids disclosed herein. In some embodiments, the nucleic acid tethering the guide nucleic acid to the surface may comprise any of the reporter nucleic acids described herein. In some embodiments, the programmable nuclease-enzyme fusion protein may comprise any of the enzymes described herein.
In some embodiments, a catalytically inactive programmable nuclease may have no substantial nucleic acid-cleaving activity. In some embodiments, a catalytically inactive programmable nuclease may be catalytically inactive or “dead,” that is, they may bind to a target nucleic acid but not cleave the target nucleic acid. A catalytically inactive or dead programmable nuclease may comprise a catalytically inactive domain (e.g., inactive nuclease domain). In some instances, a catalytically inactive domain may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to an active domain of a catalytically active programmable nuclease. In some instances, the mutations may be present within a cleaving or active site of the programmable nuclease. In some instances, a catalytically inactive or dead programmable nuclease is fused to a fusion partner. In some instances, catalytically inactive may refer to an activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to a wild-type counterpart of the programmable nuclease.
In an exemplary embodiment, a target nucleic acid (19401) may bind with a programmable nuclease-split enzyme fusion protein comprising a dCas protein, an “S” subunit of a split HRP protein, and a first guide nucleic acid. In some embodiments, the dCas protein is not capable of cis or trans cleavage. The target nucleic acid (19402) may also bind with another programmable nuclease-split enzyme fusion protein (19406) comprising a dCas protein (19407), an “L” subunit of a split HRP protein (19408), and a second guide nucleic acid (19409). The first guide nucleic acid (19402) may be configured to target a first portion (19410) of the target nucleic sequence. The second guide nucleic acid (19409) may be configured to target a second portion (19411) of the target nucleic acid sequence, wherein the second portion is sufficiently close to the first portion such that the “S” subunit of the HRP (19404) and the “L” subunit of the HRP (19408) can dock together to form an activated HRP protein (19412). The activated HRP protein (19412) then may act on an enzyme substrate (19413) in solution to induce a color change.
In some embodiments, the programmable nuclease-split enzyme fusion protein may comprise any one of the programmable nucleases disclosed herein. In some embodiments, the target nucleic acid may be any one of the target nucleic acids disclosed herein. In some embodiments, the guide nucleic acids may comprise any of the guide nucleic acids disclosed herein. In some embodiments, the programmable nuclease-split enzyme fusion protein may comprise any of the enzymes described herein in split form.
In one aspect, the present disclosure provides a composition for detecting a target nucleic acid, in which the composition comprises a programmable nuclease, a guide nucleic acid, a first reporter, an enzyme, and a second reporter. In some embodiments, (a) the guide nucleic acid is configured to bind to the target nucleic acid; (b) the programmable nuclease is effective to cleave the first reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (c) the cleavage of the first reporter is effective to separate a first nucleic acid section from a second nucleic acid section thereof; (d) the first nucleic acid section is effective to activate the enzyme; and (e) the activated enzyme is effective to cleave the second reporter to produce a detectable product comprising a detection moiety. In some embodiments, the programmable nuclease is a Cas protein or fragment thereof. In some embodiments, the programmable nuclease is a Cas13. In some embodiments, the programmable nuclease is a Cas13a. In some embodiments, the programmable nuclease is a Cas13c. In some embodiments, the programmable nuclease is thermostable at elevated temperatures. In some embodiments, the enzyme is an endonuclease and the second reporter comprises a polynucleotide substrate of the enzyme. In some embodiments, the endonuclease is a NucC endonuclease. In some embodiments, the first nucleic acid section comprises adenosine residues (such as cyclic adenylate) effective to activate the NucC. In some embodiments, the second nucleic acid section comprises RNA residues, optionally wherein the RNA residues comprise a plurality of uracil residues (e.g., at least 4, 5, 6, 7, 8, 9 or 10 RNA residues, such as uracils). In some embodiments, the second nucleic acid section comprises DNA residues, optionally wherein the DNA residues comprise a plurality of thymine residues (e.g., at least 4, 5, 6, 7, 8, 9 or 10 DNA residues, such as thymines). In some embodiments, (a) the second reporter comprises a fluorescent label and a quencher, and (b) cleavage of the second reporter by the activated enzyme is effective to separate the fluorescent label from the quencher.
In some embodiments, such as when the endonuclease is NucC, the first reporter may comprise a first nucleic acid section comprising a plurality of adenosine residues and a second nucleic acid section comprising a plurality of uracil residues. In some embodiments, one or more of the plurality of adenosine residues may be modified (e.g., fluoro-adenosine, etc.) as described herein. When the target nucleic acid is a target RNA, the programmable nuclease may comprise a Cas13 nuclease which, when activated by the target RNA, may preferentially cleave the uracil residues off of the first reporter and release the first nucleic acid section comprising the adenosine residues. NucC is naturally activated by cyclic adenylate (cA3) and cleaves double-stranded DNA, thus in an exemplary embodiment the plurality of adenosine residues may be at least two or three adenosine residues and the second reporter may comprise a detection moiety (e.g., a fluorophore) and a nucleic acid comprising double-stranded DNA. In some embodiments, the second reporter may comprise a detection moiety (e.g., a fluorophore) and a quencher moiety. Cleavage of the second reporter may separate the detection moiety from the quencher moiety and generate a detectable signal as described herein.
In some embodiments, such as when the endonuclease is NucC, the composition for detecting the target nucleic acid and the target nucleic acid are reacted and analyzed at room temperature. In some embodiments, the composition for detecting the target nucleic acid and the target nucleic acid are reacted and analyzed at an elevated temperature. In some embodiments, the elevated temperature is a temperature selected from: 40° C., 45° C., 50° C., 55° C., 60° C., or 65° C.
In various aspects, provided herein are methods for detecting a target nucleic acid in a sample, the methods comprising contacting the sample with a composition, device, or system described herein. In an embodiment, the method comprises (a) contacting the sample with composition described herein; (b) cleaving the first reporter with the programmable nuclease in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid, thereby releasing the first nucleic acid section; (c) activating the enzyme with the first nucleic acid section; (d) cleaving the second reporter with the activated enzyme, thereby producing the detectable product comprising the detection moiety; and; and (e) detecting the detection moiety, thereby detecting the presence of the target nucleic acid in the sample.
Described herein are various compositions, devices, and methods for detecting a target nucleic acid, utilizing a DETECTR assay as described herein. In some embodiments, the DETECTR assay utilizes reporters comprising one or more detection moieties. One or more of the detection moieties may comprise an enzyme configured for assay signal enhancement as described herein. In some embodiments, the enzyme is horseradish peroxidase (HRP). In some embodiments, enzyme-modified reporters may be immobilized to a surface and configured to release the enzyme upon cleavage of a nucleic acid of the reporter by an activated programmable nuclease-guide complex bound to a target nucleic acid as described herein. In some embodiments, one or more enzymes are conjugated to a linker, the comprises one or more nucleic acid sections, and the linker comprises a functionality for immobilization to a support (but is not immobilized to the support at the time of cleavage by an activated programmable nuclease). In some embodiments, the reporters may comprise two or more enzymes configured for signal enhancement. In some embodiments, a reporter comprising two or more enzymes configured for signal enhancement is referred to as a poly-enzyme reporter. In some embodiments, each of the two or more enzymes configured for signal enhancement are HRP. In some embodiments, wherein the two or more enzymes configured for signal enhancement are each HRP, the reporter is referred to as a poly-HRP reporter.
Described herein are various devices for detecting a target nucleic acid, comprising: a sample interface configured to receive a sample comprising the target nucleic acid; a first reaction chamber in fluid communication with the sample interface, wherein the first reaction chamber comprises a plurality of reporters, each reporter of the plurality comprising two or more enzymes; and a surface of the first reaction chamber, wherein each reporter of the plurality of reporters is immobilized to the surface of the first reaction chamber via a nucleic acid (which may comprise or be a part of a linker or tether as described herein), and a second reaction chamber in fluid communication with the first reaction chamber, wherein the second reaction chamber comprises an enzyme substrate, and wherein cleaving the nucleic acid of the reporter by a programmable nuclease complexed with a guide nucleic acid, the guide nucleic acid being configured to bind to the target nucleic acid, may release the two or more enzymes into solution, and wherein each enzyme of the two or more enzymes may be contacted with the enzyme substrate in the second reaction chamber to generate a detectable and amplified signal indicative of a presence or absence of the target nucleic acid in the sample. In some embodiments, each enzyme of the two or more enzymes comprises HRP. In some embodiments, the reporter comprises a detection moiety comprising a streptavidin conjugated to the two or more enzymes. In some embodiments, the nucleic acid of the reporter may be immobilized to the surface via a functionality (e.g., a biotin, an acrydite, an amine, or the like) as described herein. In some embodiments, the surface of the first reaction chamber may be functionalized with a corresponding functionality (e.g., a hydrogel, carboxyl group, NHS groups, or like) as described herein. In some embodiments, the tether comprises a segment(s) of cleavable DNA, cleavable RNA, or a combination thereof. In some embodiments, the tether comprises a segment(s) of non-cleavable DNA, non-cleavable RNA, or a combination thereof. In some embodiments, the enzyme comprises HRP and the enzyme substrate comprises 3,3′,5,5′-tetramethylbenzidine (TMB). In some embodiments, the surface of the first reaction chamber comprises the inner wall of the first reaction chamber. In some embodiments, the surface of the first reaction chamber comprises a surface of a bead, wherein the bead is contained within the first reaction chamber. In some embodiments, the bead is a magnetic bead.
Described herein are various devices and methods for detecting a target nucleic acid, utilizing a DETECTR assay as described herein. In some embodiments, the DETECTR assay utilizes reporters comprising a detection moiety comprising an enzyme configured for assay signal enhancement as described herein. Non-limiting examples of enzymes for assay signal enhancement include horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, catacholase, invertase, glucose, beta-lactamase, luciferase, and complexes comprising two or more thereof (e.g., two or more of the same enzyme, or two or more different enzymes). In some embodiments, the enzyme is HRP. In some embodiments, the enzyme is a poly-HRP. In some embodiments, enzyme-modified reporters may be immobilized to a surface and configured to release the enzyme upon cleavage of a nucleic acid of the reporter by an activated programmable nuclease-guide complex bound to a target nucleic acid as described herein. In some embodiments, the reporters comprise two or more enzymes configured for signal enhancement. In some embodiments, the incorporation of the two or more enzymes configured for signal enhancement may provide for assay signal enhancement upon a single cleavage event of the reporter induced by the activated programmable nuclease. In some embodiments, the two or more enzymes comprise a group, wherein the group further comprises a protein. In some embodiments, the two or more enzymes comprise a group, wherein the group further comprises a protein, the protein comprising streptavidin (SA). In some embodiments, the group comprises two or more HRP. In some embodiments, the group comprising two or more enzymes is attached to the nucleic acid of the reporter via a Biotin-Streptavidin interaction (e.g., as shown in
In some embodiments, the reporter comprises a linker (also referred to herein as a tether). In some embodiments, the linker comprises one or more linker-sections. In some embodiments, the one or more linker sections comprises a cleavable section comprising one or more nucleic acids which may be cleaved by an activated programmable nuclease as described herein. In some embodiments, the one or more linker sections comprises cleavable and non-cleavable section, the non-cleavable section comprising one or more nucleic acids, carbon spacers, or other molecules which may not be cleaved by an activated programmable nuclease. For example,
In some embodiments, the linker comprises one or more nucleic acid sections, and the only nucleic acids in the linker consist of DNA. In some embodiments, the linker comprises one or more nucleic acid sections, and the only nucleic acids in the linker consist of RNA. In some embodiments, the linker comprises one or more sections of DNA and one or more sections of RNA (e.g., one section of each, one section of RNA between two sections of DNA, one section of DNA between two sections of RNA, or greater numbers of each). In linkers comprising DNA and RNA, the DNA and RNA may be linked (e.g., covalently linked) directly to one another, or separated by one or more intervening molecules or linkages to which both the DNA and RNA are linked (e.g., covalently linked). The type, length, and arrangement of the one or more nucleic acid sections can be selected based on the programmable nuclease(s) to be used. In some embodiments, one or more DNA sections comprise one or more poly-T sections consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more contiguous thymine nucleotides. In some embodiments, the poly-T section consists of 12 contiguous thymine nucleotides (T12) (SEQ ID NO: 127). In some embodiments, the poly-T section consists of 20 contiguous thymine nucleotides (T20) (SEQ ID NO: 126). In a linker comprising two or more poly-T sections, the poly-T sections may be of the same or different lengths (e.g., two of T12 (“T12” disclosed as SEQ ID NO: 127), a T12 (SEQ ID NO: 127) and a T20 (SEQ ID NO: 126), etc.). In some embodiments, one or more RNA sections comprise one or more poly-U sections consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more contiguous uracil nucleotides. In some embodiments, the poly-U section consists of 5 contiguous uracil nucleotides (UUUUU). In a linker comprising two or more poly-U sections, the poly-U sections may be of the same or different lengths (e.g., two of UUUUU, a UUUUU and a U8, etc.) In some embodiments, the RNA section is about or less than about 20, 15, 10, or 5 nucleotides in length. In some embodiments, a linker includes only one RNA section of fewer than 20 nucleotides (e.g., about 5 nucleotides) and is adjusted to a desired length by the inclusion of one or more DNA sections (e.g., at one end or at each end). In some embodiments, the desired length adjustment is the length of 24 DNA nucleotides (e.g., as in two T12 (“T12” disclosed as SEQ ID NO: 127) sections), 40 nucleotides (e.g., as in two T20 (“T20” disclosed as SEQ ID NO: 126) sections), a length intermediate to these (e.g., a T12 (SEQ ID NO: 127) and a T20 (SEQ ID NO: 126)), or another length as specified herein. In some embodiments, the length is adjusted to a combined length of at least about 20, 30, 40, 50, 75, 100, or 200 nucleotides. In some embodiments, the length is adjusted to a combined length of about 10 to 200 nucleotides, 20 to 100 nucleotides, or 25 to 75 nucleotides. In some embodiments, the length is adjusted to a combined length of at least about 20, 30, 40, or 50 nucleotides. In some embodiments, the length is adjusted to a combined length of at least about 30 nucleotides. In some embodiments, the one or more nucleic acid sections are single-stranded.
In some embodiments, the one or more linker sections comprise at least one hydrocarbon chain. In some embodiments, the one or more hydrocarbon chains comprise a linear chain of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 75 or more carbons. In some embodiments, a hydrocarbon chain comprises one or more units (e.g., 2, 3, 4, 5, or more units) of an 18-carbon chain. In some embodiments the hydrocarbon chain is a chain of 4 carbons (C4H8). In some embodiments the hydrocarbon chain is a chain of 8 carbons (C8H16). In some embodiments, the hydrocarbon chain is a chain of at least 36 carbons. In some embodiments, the hydrocarbon chain is a chain of at least 54-carbon chain. In a linker comprising two or more hydrocarbon chains, the hydrocarbon chains can be chains of the same or different lengths (e.g., two chains of 4 carbons, a chain of 4 carbons and a chain of 8 carbons, a chain of 36 carbons and a chain of 54 carbons, etc.). In some embodiments of a linker comprising two hydrocarbon chains separated by one or more nucleic acid sections, the two hydrocarbon chains have a combine length of about or at least about 30, 50, 75, 100, or more carbons. In some embodiments, the combined chain length is at least 75 carbons. In some embodiments, a hydrocarbon chain is located at one or more of (i) between a nucleic acid section of a linker and a solid support to which the linker is attached, (ii) between a nucleic acid section of a linker and an enzyme for signal enhancement (e.g., an HRP or poly-HRP), or (iii) between two nucleic acid sections of the same or different type. In some embodiments, the linker comprises a single nucleic acid section (e.g., an RNA section) with a hydrocarbon chain at both ends, one joining the nucleic acid section to the support at one end, and the other joining the nucleic acid section to the enzyme at the other end. In some embodiments, a linker includes only one RNA section of fewer than 20 nucleotides (e.g., about 5 nucleotides) and is adjusted to a desired length by the inclusion of one or more hydrocarbon chains (e.g., at one end or at each end). In some embodiments one or more hydrocarbon chains and one or more DNA sections are used to adjust the linker length.
In some embodiments, the reporter may comprise a functionality for binding to a solid support as described herein. In some embodiments, the reporter may comprise a functionality for binding to a surface of a solid support as described herein. In some embodiments, the surface may comprise a surface of a chamber, the surface of a particle, a bead, a polymer matrix, or other scaffold as described herein. In some embodiments, the functionality may comprise a biotin, an acrydite, an amine, or the like, or a combination thereof, as described herein.
Table 4 describes reporters as shown in
In some embodiments, the reporter may be immobilized onto a solid support as described herein. In some embodiments, the reporter may be a poly-enzyme reporter. In some embodiments, one or more of the poly-enzymes of the reporter may be HRP. In some embodiments, the poly-HRP reporter may be immobilized to a bead functionalized with a functionality such as streptavidin. A variety of suitable beads are available, non-limiting examples of which include DYNABEADS MYONE carboxylic acid magnetic beads (1 μm diameter), 1 μm PIERCE streptavidin magnetic beads, 4.5 μm DYNABEADS epoxy magnetic beads, 2.7 μm DYNABEADS epoxy magnetic beads, and 1 μm PIERCE NHS-activated magnetic beads. In some embodiments, during reporter immobilization onto beads functionalized with streptavidin, the reporter may be present at a concentration of about 0.1 nM to about 1,000 nM, for example within a range of about 0.1 nM to about 10 nM. In some embodiments, during reporter immobilization, the reporter is present at a concentration of about 0.1 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 100 nM, about 0.1 nM to about 250 nM, about 0.1 nM to about 500 nM, about 0.1 nM to about 1,000 nM, about 1 nM to about 10 nM, about 1 nM to about 100 nM, about 1 nM to about 250 nM, about 1 nM to about 500 nM, about 1 nM to about 1,000 nM, about 10 nM to about 100 nM, about 10 nM to about 250 nM, about 10 nM to about 500 nM, about 10 nM to about 1,000 nM, about 100 nM to about 250 nM, about 100 nM to about 500 nM, about 100 nM to about 1,000 nM, about 250 nM to about 500 nM, about 250 nM to about 1,000 nM, or about 500 nM to about 1,000 nM. In some embodiments, during reporter immobilization, the reporter is present at a concentration of about 0.1 nM, about 1 nM, about 10 nM, about 100 nM, about 250 nM, about 500 nM, or about 1,000 nM. In some embodiments, during reporter immobilization, the reporter is present at a concentration of at least about 0.1 nM, about 1 nM, about 10 nM, about 100 nM, about 250 nM, or about 500 nM. In some embodiments, during reporter immobilization, the reporter is present at a concentration of at most about 1 nM, about 10 nM, about 100 nM, about 250 nM, about 500 nM, or about 1,000 nM.
Described herein are various embodiments of DETECTR assays that may utilize hydrogels as a support for reporters, as described herein. In some embodiments, such hydrogels may comprise poly (ethylene glycol) diacrylate, or equivalently PEGDA or PEG-DA.
In some embodiments, reporters configured for HRP-reporter-based programmable nuclease-based detection (e.g., DETECTR) assays may be immobilized onto a solid support as described herein. In some embodiments, one or more HRP-conjugated reporters may be immobilized onto a solid support comprising a resin. In some embodiments, one or more HRP-conjugated reporters may be immobilized onto a solid support comprising an activated resin. In some embodiments, one or more HRP-conjugated reporters may be immobilized onto a solid support comprising a resin activated with N-Hydroxysuccinimide (NHS) functionalities. In some embodiments, one or more HRP-conjugated reporters are immobilized onto a solid support comprising a resin activated with NHS functionalities, wherein the reporters comprise corresponding amine functionalities as described herein. In some embodiments, one or more HRP-conjugated reporters are immobilized onto a solid support comprising a resin activated with NHS functionalities, wherein the reporters comprise a protein, for example an enzyme such as HRP. In some embodiments, one or more reporters are configured for HRP-based DETECTR assays and may be immobilized onto one or more solid support comprising a resin activated with NHS functionalities, wherein the reporters comprise an HRP and an amine functionality connected by a linker as described herein. In some embodiments, the resin may comprise agarose.
In some embodiments, reporters configured for poly enzyme reporter-based DETECTR assays may be immobilized onto solid-supports. In some embodiments, reporters configured for poly HRP reporter-based DETECTR assays may be immobilized onto solid-supports comprising beads. In some embodiments, the beads are functionalized with streptavidin or NHS. In some embodiments, the poly HRP-reporters are immobilized onto the beads at a concentration of about 0.01 nM to about 0.5 μM, such as about 0.1 nM to about 250 nM, about 0.5 nM to about 100 nM, or about 1 nM to about 50 nM. In some embodiments, poly-HRP reporter molecules are immobilized at a concentration of about 10 nM. In some embodiments, mono-HRP reporter molecules are immobilized at a concentration of about 1 nM to about 50 μM, about 10 nM to about 25 μM, about 50 nM to about 10 μM, or about 100 nM to about 5 μM. In some embodiments, mono-HRP reporter molecules are immobilized at a concentration of about 1 μM. In some embodiments, HRP-DETECTR reagents are added to the beads functionalized with reporters, wherein the % solid content (e.g., concentration) of the beads is about 1 to about 50 mg/mL (e.g., about 5 to about 25, or about 10 mg/mL). In some embodiments, the reporter-functionalized beads are used at a concentration of about 10 mg/mL.
In various aspects, the present disclosure provides methods of using the compositions, devices, and systems disclosed herein to detect a target nucleic acid. In some embodiments, the method comprises (a) contacting the sample with a composition disclosed herein to produce a reaction fluid, wherein (i) the linker is conjugated to the support, (ii) the composition further comprises a guide nucleic acid configured to bind to the target nucleic acid, and (iii) the programmable nuclease cleaves the linker in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (b) separating the reaction fluid from the support; (c) contacting the separated reaction fluid with a substrate of the one or more enzymes, wherein presence of the one or more enzymes in the fluid produces a detectable signal from the enzyme substrate; and (d) detecting the detectable signal. In some embodiments, the method comprises (a) contacting the sample with a composition disclosed herein to produce a reaction fluid, wherein (i) the composition further comprises a programmable nuclease and a guide nucleic acid, (ii) the guide nucleic acid is configured to bind to the target nucleic acid, and (iii) the programmable nuclease cleaves the linker in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (b) contacting the reaction fluid with a plurality of beads, wherein each bead reacts with a plurality of the functionalities, thereby immobilizing the functionalities to the beads; (c) separating the reaction fluid from the beads; (d) contacting the separated reaction fluid with a substrate of the one or more enzymes, wherein presence of the one or more enzymes in the fluid produces a detectable signal from the enzyme substrate; and (e) detecting the detectable signal.
In some embodiments, the method further comprises concentrating the one or more enzymes prior to contacting the one or more enzymes with the substrate, wherein the concentrating comprises separating the one or more enzymes from at least a portion of the separated reaction fluid. For example, the enzyme may be captured to a support, such as a polymer matrix. The enzyme may be retained on the polymer matrix having a volume smaller than the reaction fluid applied to the matrix, thereby concentrating the enzyme. The enzyme may be reacted with a substrate while complexed to the matrix, or may be eluted in a fluid for subsequent reaction with the substrate. In embodiments where the enzyme is reacted to the substrate while complexed to the matrix, the substrate solutions comprising the substrate may be added serially (e.g., 2, 3, 4, 5, or more times), which may then be combined to detect enzymatic activity. In some embodiments, the enzyme comprises a horseradish peroxidase (HRP).
In some cases, the reporters are immobilized to the surface by attachment to the non-naturally occurring guide nucleic acid, a linkage immobilizing the guide nucleic acid to a surface, or both. In some instances, at least one of the reporters is immobilized to the surface by attachment to an end of the non-naturally occurring guide nucleic acid that is distal to the linkage. For example, at least one of the reporters can be immobilized by attaching to the 3′ end of a spacer region of the guide nucleic acid. In some cases, at least one of the reporters is immobilized to the surface by attachment to the linkage. For example, at least one of the reporters can be immobilized by attaching to a tether. In some embodiments, a plurality of reporters (e.g., at least 2, 3, 4, 5, 10, 15, or more) are attached to the linkage. In some embodiments, reporters are attached to both the linkage and an end of the guide nucleic acid. In some cases, a reporter comprises a detection moiety. In some cases, a reporter comprises an enzyme, such as HRP, as shown in
Methods of Making Polymer Matrices with Immobilized Reporters
Co-polymerization of the reporter into the hydrogel may result in a higher density of reporter/unit volume or reporter/unit area than other immobilization methods utilizing surface immobilization (e.g., onto beads). Co-polymerization of the reporter into the hydrogel may result in less undesired release of the reporter (e.g., during an assay, a measurement, or on the shelf), and thus may cause less background signal, than other immobilization strategies (e.g., conjugation to a pre-formed hydrogel, bead, etc.). In at least some instances this may be due to better incorporation of reporters into the hydrogel as a co-polymer and fewer “free” reporter molecules retained on the hydrogel via non-covalent interactions or non-specific binding interactions.
In some embodiments, the plurality of oligomers and the plurality of polymerizable oligomers may comprise an irregular or non-uniform mixture. The irregularity of the mixture of polymerizable oligomers and unfunctionalized oligomers may allow pores to form within the hydrogel (i.e., the unfunctionalized oligomers may act as a porogen). For example, the irregular mixture of oligomers may result in phase separation during polymerization that allows for the generation of pores of sufficient size for programmable nucleases to diffuse into the hydrogel and access internal reporter molecules. The relative percentages and/or molecular weights of the oligomers may be varied to vary the pore size of the hydrogel. For example, pore size may be tailored to increase the diffusion coefficient of the programmable nucleases.
In some embodiments, the functional groups attached to the reporters may be selected to preferentially incorporate the reporters into the hydrogel matrix via covalent binding at the functional group versus other locations along the nucleic acid of the reporter. In some embodiments, the functional groups attached to the reporters may be selected to favorably transfer free radicals from the functionalized ends of polymerizable oligomers to the functional group on the end of the reporter (e.g., 5′ end), thereby forming a covalent bond and immobilizing the reporter rather than destroying other parts of the reporter molecules.
In some embodiments, the polymerizable composition may further comprise one or more polymerizable nucleic acids. In some embodiments, the polymerizable nucleic acids may comprise guide nucleic acids (e.g., guide nucleic acids 15003a, 15003b, or 15003c shown in
In some embodiments, the oligomers may form a polymer matrix comprising a hydrogel. In some embodiments, the oligomers may comprise poly (ethylene glycol) (PEG), poly (siloxane), poly (hydroxyethyl acrylate, poly (acrylic acid), poly (vinyl alcohol), poly (butyl acrylate), poly (2-ethylhexyl acrylate), poly (methyl acrylate), poly (ethyl acrylate), poly (acrylonitrile), poly (methyl methacrylate), poly (acrylamide), poly (TMPTA methacrylate), chitosan, alginate, or the like, or any combination thereof. One of ordinary skill in the art will recognize that the oligomers may comprise any oligomer or mix of oligomers capable of forming a hydrogel.
In some embodiments, the oligomers may comprise polar monomers, nonpolar monomers, protic monomers, aprotic monomers, solvophobic monomers, or solvophillic monomers, or any combination thereof.
In some embodiments, the oligomers may comprise a linear topology, branched topology, star topology, dendritic topology, hyperbranched topology, bottlebrush topology, ring topology, catenated topology, or any combination thereof. In some embodiments, the oligomers may comprise 3-armed topology, 4-armed topology, 5-armed topology, 6-armed topology, 7-armed topology, 8-armed topology, 9-armed topology, or 10-armed topology.
In some embodiments, the oligomers may comprise at least about 2 monomers, at least about 3 monomers, at least about 4 monomers, at least about 5 monomers, at least about 6 monomers, at least about 7 monomers, at least about 8 monomers, at least about 9 monomers, at least about 10 monomers, at least about 20 monomers, at least about 30 monomers, at least about 40 monomers, at least about 50 monomers, at least about 60 monomers, at least about 70 monomers, at least about 80 monomers, at least about 90 monomers, at least about 100 monomers, at least about 200 monomers, at least about 300 monomers, at least about 400 monomers, at least about 500 monomers, at least about 600 monomers, at least about 700 monomers, at least about 800 monomers, at least about 900 monomers, at least about 1000 monomers, at least about 2000 monomers, at least about 3000 monomers, at least about 4000 monomers, at least about 5000 monomers, at least about 6000 monomers, at least about 7000 monomers, at least about 8000 monomers, at least about 9000 monomers, at least about 10000 monomers, at least about 20000 monomers, at least about 30000 monomers, at least about 40000 monomers, at least about 50000 monomers, at least about 60000 monomers, at least about 70000 monomers, at least about 80000 monomers, at least about 90000 monomers, or at least about 100000 monomers.
In some embodiments, the oligomers may comprise a homopolymer, a copolymer, a random copolymer, a block copolymer, an alternative copolymer, a copolymer with regular repeating units, or any combination thereof.
In some embodiments, the oligomers may comprise 1 type of monomer, 2 types of monomers, 3 types of monomers, 4 types of monomers, 5 types of monomers, 6 types of monomers, 7 types of monomers, 8 types of monomers, 9 types of monomers, or 10 types of monomers.
The polymerizable oligomers may comprise any of the oligomers described herein. In some embodiments, the polymerizable oligomers may comprise one or more functional groups. In some embodiments, the functional group may comprise an acrylate group, N-hydroxysuccinimide ester group, thiol group, carboxyl group, azide group, alkyne group, an alkene group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of functional groups may be used to functionalize oligomers into polymerizable oligomers depending on the desired properties of the polymerizable oligomers.
In some embodiments, the polymerizable oligomers may form a polymer matrix comprising a hydrogel. In some embodiments, the polymerizable oligomers may comprise PEG, poly (siloxane), poly (hydroxyethyl acrylate, poly (acrylic acid), poly (vinyl alcohol), or any combination thereof. One of ordinary skill in the art will recognize that the set of polymerizable oligomers may comprise any polymer capable of forming a hydrogel.
In some embodiments, the set of polymerizable oligomers comprises polar monomers, nonpolar monomers, protic monomers, aprotic monomers, solvophobic monomers, or solvophillic monomers.
In some embodiments, the set of polymerizable oligomers comprises a linear topology, branched topology, star topology, dendritic topology, hyperbranched topology, bottlebrush topology, ring topology, catenated topology, or any combination thereof. In some embodiments, the set of polymerizable oligomers comprises 3-armed topology, 4-armed topology, 5-armed topology, 6-armed topology, 7-armed topology, 8-armed topology, 9-armed topology, or 10-armed topology.
In some embodiments, the set of polymerizable oligomers comprises at least about 2 monomers, at least about 3 monomers, at least about 4 monomers, at least about 5 monomers, at least about 6 monomers, at least about 7 monomers, at least about 8 monomers, at least about 9 monomers, at least about 10 monomers, at least about 20 monomers, at least about 30 monomers, at least about 40 monomers, at least about 50 monomers, at least about 60 monomers, at least about 70 monomers, at least about 80 monomers, at least about 90 monomers, at least about 100 monomers, at least about 200 monomers, at least about 300 monomers, at least about 400 monomers, at least about 500 monomers, at least about 600 monomers, at least about 700 monomers, at least about 800 monomers, at least about 900 monomers, at least about 1000 monomers, at least about 2000 monomers, at least about 3000 monomers, at least about 4000 monomers, at least about 5000 monomers, at least about 6000 monomers, at least about 7000 monomers, at least about 8000 monomers, at least about 9000 monomers, at least about 10000 monomers, at least about 20000 monomers, at least about 30000 monomers, at least about 40000 monomers, at least about 50000 monomers, at least about 60000 monomers, at least about 70000 monomers, at least about 80000 monomers, at least about 90000 monomers, or at least about 100000 monomers. As used herein, “about” may mean plus or minus 1 monomer, plus or minus 10 monomers, plus or minus 100 monomers, plus or minus 1000 monomers, plus or minus 10000 monomers, or plus or minus 100000 monomers.
In some embodiments, the set of polymerizable oligomers comprises a homopolymer, a copolymer, a random copolymer, a block copolymer, an alternative copolymer, a copolymer with regular repeating units, or any combination thereof.
In some embodiments, the set of polymerizable oligomers comprises 1 type of monomer, 2 types of monomers, 3 types of monomers, 4 types of monomers, 5 types of monomers, 6 types of monomers, 7 types of monomers, 8 types of monomers, 9 types of monomers, or 10 types of monomers.
In some embodiments, the polymerizable composition may comprise a mix of unfunctionalized or unmodified oligomers and polymerizable oligomers as described herein. In some embodiments, the unfunctionalized or unmodified oligomers may act as porogens to generate pores within the polymer matrix.
The polymerizable reporters may comprise any of the reporters described herein. In some embodiments, the set of polymerizable reporters may comprise one or more functional groups. In some embodiments, the functional group may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5′ thiol modifier, a 3′ thiol modifier, an amine group, a I-Linker™ group, methacryl group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of functional groups may be used with the set of polymerizable reporters depending on the desired properties of the polymerizable reporters.
In some embodiments, the set of initiators may comprise one or more photoinitiators or thermal initiators. In some embodiments, the set of initiators may comprise cationic initiators, anionic initiators, or radical initiators. In some embodiments, the set of initiators may comprise AIBN, AMBN, ADVN, ACVA, dimethyl 2,2′-azo-bis (2methylpropionate), AAPH, 2,2′-azobis [2-(2-imidazolin-2-yl)-propane] dihydrochloride, TBHP, cumene hydroperoxide, di-tert-butyl peroxide, dicumyl peroxide, BPO, dicyandamide, cyclohexyl tosylate, diphenyl (methyl) sulfonium tetrafluoroborate, benzyl (4-hydroxyphenyl)-methylsulfonium hexafluoroantimonate, (4-hydroxyphenyl) methyl-(2-methylbenzyl) sulfonium hexafluoroantimonate, camphorquinone, acetophenone, 3-acetophenol, 4-acetophenol, benzophenone, 2-methylbenzophenone, 3-methylbenzophenone, 3-hydroxybenzophenone, 3,4-dimethylbenzophenone, 4-hydroxybenzophenone, 4-benzoylbenzoic acid, 2-benzoylbenzoic acid, methyl 2-benzoylbenzoate, 4,4′-dihydroxybenzophenone, 4-(dimethylamino)-benzophenone, 4,4′-bis (dimethylamino)-benzophenone, 4,4′-bis (diethylamino)-benzophenone, 4,4′-dichlorobenzophenone, 4-(p-tolylthio) benzophenone, 4-phenylbenzophenone, 1,4-dibenzoylbenzene, benzil, 4,4′-dimethylbenzil, p-anisil, 2-benzoyl-2-propanol, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, 1-benzoylchclohexanol, benzoin, anisoin, benzoin methyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin isobutyl ether, o-tosylbenzoin, 2,2-diethoxyacetophenone, benzil dimethylketal, 2-methyl-4′-(methylthio)-2-morpholinopropiophenone, 2-benzyl-2-(dimethylamino)-4′-morpholinobutyrophenone, 2-isonitrosopropiophenone, anthraquinone, 2-ethylantraquinone, sodium anthraquinone-2-sulfonate monohydrate, 9,10-phenanthrenequinone, 9,10-phenanthrenequinone, dibenzosuberenone, 2-chlorothioxanthone, 2-isopropylthioxanthone, 2,4-diethylthioxanthen-9-one, 2,2′bis (2-chlorophenyl)-4,4′,5,5′-tetraphenyl-1,2′-biimidazole, diphenyl (2,4,6-trimethyl-benzoyl) phosphine oxide, phenylbis (2,4,6-trimethyl-benzoyl) phosphine oxide, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, diphenyliodonium trifluoromethanesulfonate, diphenyliodonium hexafluorophosphate, diphenyliodonium hexafluoroarsenate, bis (4-tert-butylphenyl)-iodonium triflate, bis (4-tert-butylphenyl) iodonium hexafluorophosphate, 4-isopropyl-4′-methyl-diphenyliodonium tetrakis (pentafluorophenyl) borate, [4-[(2-hydroxytetradecyl)-oxy]phenyl]phenyliodonium hexafluoroantimonate, bis [4-(tert-butyl) phenyl]-iodonium tetra (nonafluoro-tert-butoxy) aluminate, cyclopropyldiphenylsulfonium tetrafluoroborate, triphenylsulfonium bromide, triphenylsulfonium tetrafluoroborate, tri-p-tolylsulfonium triflate, tri-p-tolylsulfonium hexafluorophosphate, 4-nitrobenzenediazonium tetrafluoroborate, 2-(4-methoxyphenyl)-4,6-bis (trichloromethyl)-1,3,5-triazine, 2-(1,3-benzodioxol-5-yl)-4,6-bis (trichloromethyl)-1,3,5-triazine, 2-(4-methoxystyryl)-4,6-bis (trichloromethyl)-1,3,5-triazine, 2-(3,4-dimethoxystyryl)-4,6-bis (trichloromethyl)-1,3,5-triazine, 2-[2-(Furan-2-yl) vinyl]-4,6-bis (trichloromethyl)-1,3,5-triazine, 2-[2-(5-methylfuran-2-yl) vinyl]-4,6-bis (trichloromethyl)-1,3,5-triazine, 2-(9-oxoxanthen-2-yl) proprionic acid 1,5,7-triazabicyclo[4.4.0]dec-5-ene salt, 2-(9-oxoxanthen-2-yl) proprionic acid 1,5-diazabicyclo[4.3.0]non-5-ene salt, 2-(9-oxoxanthen-2-yl) proprionic acid 1,8-diazabicyclo [5.4.0]-undec-7-ene salt, acetophenone O-benzoyloxime, 2-nitrobenzyl cyclohexylcarbamate, 1,2-bis (4-methoxyphenyl)-2-oxoethyl cyclohexylcarbamate, tert-amyl peroxybenzoate, 4,4-azobis (4-cyanovaleric acid), 1,1′-azobis (cyclohexanecarbonitrile), 2,2′-azobisisobutyronitrile, benzoyl peroxide, 2,2-bi (tert-butylperoxy) butane, 1,1-bis (tert-butylperoxy) cyclohexane, 2,5-bis (tert-butylperoxy)-2,5-dimethylhexane, bis (1-(tert-butylperoxy)-1-methylethyl) benzene, 1,1-bis (tert-butylperoxy)-3,3,5-trimethylcyclohexane, tert-butyl hydroperoxide, tert-butyl peracetate, tert-butyl peroxide, tert-butyl peroxybenzoate, tert-butylperoxy isopropyl carbonate, cumene hydroperoxide, cyclohexanone peroxide, dicumyl peroxide, lauroyl peroxide, 2,4-pentanedione peroxide, peracetic acid, potassium persulfate, 2-Hydroxy-2-methylpropiophenone, or any combination thereof. One of ordinary skill in the art will recognize that a variety of initiators may be used depending on the desired reaction conditions and chemistries.
In some embodiments, the initiation stimulus is UV light. In some embodiments, the initiation stimulus is UV light through a photomask. In some embodiments, the initiation stimulus is heat.
In some embodiments, the hydrogel may comprise a circular cross-sectional shape, a rectangular cross-sectional shape, a star cross-sectional shape, a dollop shape, an amorphous shape, or any shape of interest, or any combination thereof (e.g., as shown in
In some embodiments, a mask may be used to shape the initiation stimulus deposition on the polymerizable components (e.g., oligomers, etc.) and thereby shape the resulting polymer matrix. In some embodiments, the mask may comprise a circular shape, a rectangular shape, a star shape, a dollop shape, an amorphous shape, or any shape of interest, or any combination thereof.
Hydrogel Compositions with Immobilized Reporters
In some embodiments, the composition may comprise a hydrogel (15001) comprising (a) a polymer network comprising covalently bound oligomers co-polymerized with reporters (15002) to covalently bind and immobilize the reporters to said network, and (b) immobilized programmable nuclease complexes covalently bound to said network (e.g., via co-polymerization or after reporter-immobilized polymer formation), wherein said programmable nuclease complexes may comprise a programmable nuclease (15004) and a guide nucleic acid (15003). In some embodiments, the guide nucleic acid (15003) and/or the programmable nuclease (15004) may be immobilized to or in the hydrogel as described herein (e.g., during or after formation of the hydrogel).
In some embodiments, the network of covalently bound oligomers may comprise a network formed by polymerizing one or more PEG species. In some embodiments, the network of covalently bound oligomers may comprise a network formed by polymerizing PEG comprising acrylate functional groups. In some embodiments, the acrylate functional groups may be PEG end groups. In some embodiments, the network may be formed by polymerizing PEG comprising acrylate functional groups with unmodified PEG. The molecular weight of the acrylate-modified PEG (e.g., PEG-diacrylate) and the unmodified PEG may be the same or different.
In some embodiments, the network of covalently bound oligomers may comprise a network formed from polymerizing one or more PEG species, wherein each PEG species may comprise a linear topology, branched topology, star topology, dendritic topology, hyperbranched topology, bottlebrush topology, ring topology, catenated topology, or any combination thereof. In some embodiments, the network of covalently bound oligomers may comprise a network formed from polymerizing one or more PEG species comprising a 3-armed topology, a 4-armed topology, a 5-armed topology, a 6-armed topology, a 7-armed topology, a 8-armed topology, a 9-armed topology, or a 10-armed topology.
In some embodiments, the immobilized reporter may comprise a reporter molecule covalently bound to a linker molecule, wherein the linker molecule is covalently bound to the hydrogel (e.g., via co-polymerization with the oligomers as described herein). In some embodiments, the linker molecule may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5′ thiol modifier, a 3′ thiol modifier, an amine group, a I-Linker™ group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of linker molecules may be used.
In some cases, the immobilized guide nucleic acid may comprise a guide nucleic acid covalently bound to a linker molecule, wherein the linker molecule is covalently bound to the hydrogel. In some embodiments, the linker molecule may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5′ thiol modifier, a 3′ thiol modifier, an amine group, a I-Linker™ group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of linker molecules may be used.
In some cases, the immobilized programmable nuclease may comprise a programmable nuclease covalently bound to a linker molecule, wherein the linker molecule is covalently bound to the hydrogel. In some embodiments, the linker molecule may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5′ thiol modifier, a 3′ thiol modifier, an amine group, a I-Linker™ group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of linker molecules may be used.
Methods of Using Hydrogels with Immobilized Reporters
Any of the methods described herein may utilize hydrogels (14901) with immobilized reporters (14902) for target detection assays. In some embodiments, the hydrogel (14901) comprises (a) a network of covalently bound oligomers (14903) and (b) immobilized reporters (14902) covalently bound to said network (14903) as shown in
Any of the multiplexing methods described herein may utilize hydrogels (15001a, 15001b, 15001c, etc.) with immobilized reporters (15002) for multiplexed target detection assays. In some embodiments, each hydrogel (15001a, 150001b, 15001c, etc.) may comprise (a) a polymer network of covalently bound oligomers co-polymerized with reporters (15002) to covalently bind and immobilize the reporters to said network, and (b) one or more immobilized programmable nuclease complexes covalently bound to said network as shown in
Devices Comprising Hydrogels with Immobilized Reporters
Any of the systems or devices described herein may comprise one or more hydrogels with immobilized reporters.
In some embodiments, the systems and devices described herein may comprise a plurality of hydrogels each comprising reporter molecules (e.g., in order to facilitate multiplexing and/or improve signal). In some embodiments, a first hydrogel may comprise a shape different from a shape of a second hydrogel. In some embodiments, the first hydrogel may comprise a plurality of first reporter molecules different from a plurality of second reporter molecules of the second hydrogel. In some embodiments, the reporters are the same in the first and second hydrogels. In some embodiments, the first hydrogel may comprise a circular shape, a square shape, a star shape, or any other shape distinguishable from a shape of the second hydrogel. In some embodiments, the plurality of first reporter molecules may each comprise a sequence cleavable by a programmable nuclease complex comprising a first programmable nuclease and a first guide nucleic acid. In some embodiments, the plurality of second reporter molecules may each comprise a sequence not cleavable by the first programmable nuclease complex.
Any of the systems or devices described herein may comprise a plurality of hydrogels each comprising reporter molecules. For example, a first hydrogel may comprise a plurality of first reporter molecules different from a plurality of second reporter molecules of a second hydrogel. In some embodiments, the plurality of first reporter molecules may each comprise a first fluorescent moiety, wherein the first fluorescent moiety is different than second fluorescent moieties of in each of the plurality of second reporter molecules. In some embodiments, the plurality of first reporter molecules may each comprise a sequence cleavable by a first programmable nuclease complex comprising a first programmable nuclease and a first guide nucleic acid. In some embodiments, the plurality of second reporter molecules may each comprise a sequence cleavable by a second programmable nuclease complex comprising a second programmable nuclease and a second guide nucleic acid.
Any of the systems or devices described herein may comprise at least about 2 hydrogels, at least about 3 hydrogels, at least about 4 hydrogels, at least about 5 hydrogels, at least about 6 hydrogels, at least about 7 hydrogels, at least about 8 hydrogels, at least about 9 hydrogels, at least about 10 hydrogels, at least about 20 hydrogels, at least about 30 hydrogels, at least about 40 hydrogels, at least about 50 hydrogels, at least about 60 hydrogels, at least about 70 hydrogels, at least about 80 hydrogels, at least about 90 hydrogels, at least about 100 hydrogels, at least about 200 hydrogels, at least about 300 hydrogels, at least about 400 hydrogels, at least about 500 hydrogels, at least about 600 hydrogels, at least about 700 hydrogels, at least about 800 hydrogels, at least about 900 hydrogels, at least about 1000 hydrogels,
Any of the systems or devices described herein may comprise one or more compartments, chambers, channels, or locations comprising the one or more hydrogels. In some embodiments, two or more of the compartments may be in fluid communication, optical communication, thermal communication, or any combination thereof with one another. In some embodiments, two or more compartments may be arranged in a sequence. In some embodiments, two or more compartments may be arranged in parallel. In some embodiments, two or more compartments may be arranged in sequence, parallel, or both. In some embodiments, one or more compartments may comprise a well. In some embodiments, one or more compartments may comprise a flow strip. In some embodiments, one or more compartments may comprise a heating element.
In some embodiments, the device may be a handheld device. In some embodiments, the device may be point-of-need device. In some embodiments, the device may comprise any one of the device configurations described herein. In some embodiments, the device may comprise one or more parts of any one of the device configurations described herein.
Any of the methods described herein may comprise amplifying a detection signal using a positive feedback system.
A single first target nucleic acid can lead to the release of a plurality of secondary target-specific guide nucleic acids and the generation of the plurality of second activated programmable nuclease complexes as described herein. Then each second activated programmable nuclease complex can lead to the generation of another plurality of secondary target-specific guide nucleic acids and another plurality of second activated programmable nuclease complexes. And so on. The second activated programmable nuclease complexes can generate additional signal beyond that of the first activated programmable nuclease complex alone as described herein. Therefore, the detection of the single first target nucleic acid can activate a positive feedback loop for amplifying its signal.
In some aspects, provided herein are compositions for amplifying a detection signal using a positive feedback system. In some embodiments, a composition may comprise: (a) a first set of programmable nucleases (15101) each comprising a first guide nucleic acid (15102), wherein each programmable nuclease in the first set of programmable nucleases is configured to bind with a first target nucleic acid (15103) and then trans-cleave a plurality of nucleic acids comprising a first sequence; (b) a plurality of secondary target-specific guide nucleic acids (15104) each comprising a nucleic acid tether (15106) comprising the first sequence; (c) a second set of programmable nucleases (15101) each configured to bind with a secondary target-specific guide nucleic acid which is configured to bind with a second target nucleic acid and then cleave a plurality of nucleic acids comprising a second sequence; a plurality of second target nucleic acids (15108) each comprising the second sequence.
In some embodiments, the first programmable nuclease (15101) may be free in solution, as illustrated in
In some embodiments, one or more components of the composition may be immobilized on a substrate (e.g., 15105, 15109).
In some embodiments, the substrate (15105, 15109) may comprise a hydrogel, as illustrated in
In some embodiments, the secondary target-specific guide nucleic acids (15104) may be immobilized to a substrate (15105, 15109). In some embodiments, the secondary target-specific guide nucleic acids (15104) may be immobilized with a single stranded nucleic acid tether (15106). In some embodiments, the secondary target-specific guide nucleic acids (15104) may be free in solution. In some embodiments, the secondary target-specific guide nucleic acids (15104) may comprise reporters. In some embodiments, the secondary target-specific guide nucleic acids (15104) may comprise detection moieties (15111).
In some embodiments, the second target nucleic acids (15108) may be immobilized on a substrate (15105, 15109). In some embodiments, the second target nucleic acids (15108) may be free in solution. In some embodiments, the second target nucleic acids (15108) may comprise reporters. In some embodiments, the second target nucleic acids (15108) may comprise detection moieties (15111).
In some embodiments, the compositions for amplifying a detection signal using a positive feedback system can be described step-wise as shown in
The programmable nucleases may comprise any of the programmable nucleases described herein. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is a Cas9 enzyme. In some embodiments, the nuclease is a mutant Cas9 enzyme. In some embodiments, the nuclease is an engineered Cas9 enzyme. In some embodiments, the nuclease is a Cas12 enzyme. In some embodiments, the nuclease is a Cas13 enzyme. In some embodiments, the nuclease is a Cas14 enzyme. In some embodiments, the nuclease is a CasPhi enzyme.
In some embodiments, the first and second programmable nucleases are the same. In some embodiments, the first and second programmable nucleases are different. In some embodiments, the programmable nucleases carry out cis cleavage. In some embodiments, the programmable nucleases carry out trans cleavage.
In some embodiments, cleaving by a programmable nuclease activates a reporter. In some embodiments, cleaving by a programmable nuclease activates (e.g., releases, unquenches, etc.) a detection moiety. In some embodiments, cleaving a nucleic acid tether activates a reporter. In some embodiments, cleaving a second target nucleic acid activates a reporter.
Any of the devices described herein may be configured for amplifying a detection signal using a positive feedback system. In some embodiments, a device may comprise one or more compartments configured to: (a) bind a first nuclease (15101) with a first guide nucleic acid (15102) and a first target nucleic acid (15103) to generate a first complex; (b) release one or more second guide nucleic acids (15104) each comprising a nucleic acid tether (15106) by cleaving the nucleic acid tether(s) (15106) with the first complex; (c) bind the second guide nucleic acids (15104) each with a second nuclease (15101) and a second target nucleic acid (15108) to generate a plurality of second complexes; (d) cleave a plurality of reporters with the first and second complexes as described herein. Additional second complexes may be formed by further cleavage by the first and second complexes as described herein.
In some embodiments, a device may comprise one or more compartments comprising: (a) a first set of nucleases (15101) each comprising a first guide nucleic acid (15402), wherein each nuclease in the first set of nucleases is configured to bind with a first target nucleic acid (15103) and then cleave a plurality of nucleic acids comprising a first sequence; (b) a plurality of secondary target-specific guide nucleic acids (15104)) each comprising a nucleic acid tether (15106) comprising the first sequence; (c) a second set of nucleases (15101) each configured to bind with the secondary target-specific guide nucleic acid (15104) and a second target nucleic acid (15108) and then cleave a plurality of nucleic acids; (d) a plurality of second target nucleic acids (15108).
In some embodiments, one or more compartments may be in fluid communication, optical communication, thermal communication, or any combination thereof with one another. In some embodiments, one or more compartments may be arranged in a sequence. In some embodiments, one or more compartments may be arranged in parallel. In some embodiments, one or more compartments may be arranged in sequence, parallel, or both. In some embodiments, one or more compartments may comprise a well. In some embodiments, one or more compartments may comprise a flow strip. In some embodiments, one or more compartments may comprise a heating element.
In some embodiments, the device may be a handheld device. In some embodiments, the device may be point-of-need device. In some embodiments, the device may comprise any one of the device configurations described in this disclosure. In some embodiments, the device may comprise one or more parts of any one of the device configurations described herein.
Assay Compositions with Thermostable Inorganic Pyrophosphatase
Any of the systems, methods, or devices described herein may comprise using thermostable inorganic pyrophosphatase (TIPP). In some embodiments, a composition for an assay may comprise TIPP. In some embodiments, the composition may comprise about 0.5 enzyme unit (U) per 10 μL of solution. In some embodiments, the composition may comprise at least about 0.1 U per 10 μL of solution. In some embodiments, the composition may comprise at most about 2 U per 10 μL of solution. In some embodiments, the composition may comprise at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 U per 10 μL of solution. In some embodiments, the composition may comprise at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 U per 10 μL of solution.
In some embodiments, TIPP may improve the signal to noise ratio of a programmable nuclease-based detection reaction. In some embodiments, TIPP may improve overall signal (e.g., fluorescence of a cleaved reporter as shown in
In some cases, the TIPP enzyme may be present in the HotPot reaction mix at a concentration of 0.125 Units, 0.5 Units, 0.25 Units, 1.0 Units, 2.0 Units, 2.5 Units, or 4 Units per discrete reaction volume. In some cases, the buffer may comprise the TIPP signal enhancement reagent. Conversely, the TIPP signal enhancement reagent may be provided separately (e.g., in a separate vial in a kit) and added to a reaction mixture at a desired timepoint.
In some cases, a buffer may comprise a catalytic reagent for signal improvement or enhancement. In some cases, the catalytic reagent may enhance signal generation via hydrolysis of inorganic pyrophosphates. In some cases, the catalytic reagent may enhance signal generation via enhancement of DNA replication. In some cases, the catalytic reagent may enhance signal amplification via revival of Mg+2 ions in the buffer solution which may otherwise be taken up by the phosphates produced from usage of dNTPs during the LAMP or NEAR reaction (or other isothermal or thermocycling replication reaction described herein). In some cases, the catalytic reagent may enhance signal generation by reviving the concentration of Mg+2 ions in the buffer thereby enhancing the function of the Cas nuclease effector enzyme. In some cases, the catalytic reagent for signal improvement may be an enzyme. In some cases, the catalytic reagent for signal improvement may be a Thermostable Inorganic Pyrophosphatase (TIPP).
In some embodiments, a target nucleic acid may be amplified using a nicking enzyme amplification reaction (NEAR). NEAR may be used to amplify a region of a nucleic acid comprising a target nucleic acid. NEAR may comprise a forward primer and a reverse primer that at least partially anneals to complementary strands of a target nucleic acid 3′ of the region to be amplified. The forward primer and the reverse primer may comprise a stabilizing region that is not complementary to the target sequence. The forward primer and the reverse primer may comprise recognition regions that may be nicked by a nicking enzyme. A polymerase may polymerize a nucleic acid 5′ to 3′ from the 3′ end of the forward primer or the 3′ end of the reverse primer, using the strand to which the primer is annealed as a template, resulting in a double stranded nucleic acid product or amplicon. The newly synthesized strand may comprise a restriction site and may then serve as a template. The resulting double stranded nucleic acid amplicon may comprise nicking sites in both strands. A nicking enzyme may nick a single strand of the double stranded product or amplicon. The polymerase may polymerize a nucleic acid 5′ to 3′ from the 3′ end of the nucleic acid amplicon 5′ of the nick. The process may be repeated, thereby amplifying the target nucleic acid.
As described herein, a target nucleic acid may be detected using a DNA-activated programmable RNA nuclease (e.g., a Cas13), a DNA-activated programmable DNA nuclease (e.g., a Cas12), or an RNA-activated programmable RNA nuclease (e.g., a Cas13) and other reagents disclosed herein (e.g., RNA components). The target nucleic acid may be detected using DETECTR, as described herein. The target nucleic acid may be an RNA, reverse transcribed RNA, DNA, DNA amplicon, amplified DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. In some embodiments, the target nucleic acid is amplified prior to or concurrent with detection. In some embodiments, the target nucleic acid is reverse transcribed prior to amplification. The target nucleic acid may be amplified via NEAR of a target nucleic acid sequence. In some embodiments, the nucleic acid is amplified using NEAR coupled with reverse transcription (RT-NEAR). The NEAR amplification may be performed independently, or the NEAR amplification may be coupled to DETECTR for detection of the target nucleic acid. The RT-NEAR amplification may be performed independently, or the RT-NEAR amplification may be coupled to DETECTR for detection of the target nucleic acid. The DETECTR reaction may be performed using any method consistent with the methods disclosed herein.
NEAR may be performed as an isothermal reaction, for example NEAR may be performed at from about 37° C. to about 42° C. In some embodiments, NEAR may be performed at from about 15° C. to about 60° C., from about 15° C. to about 55° C., from about 15° C. to about 50° C., from about 15° C. to about 45° C., from about 15° C. to about 40° C., from about 15° C. to about 35° C., from about 15° C. to about 30° C., from about 15° C. to about 25° C., from about 15° C. to about 20° C., from about 20° C. to about 60° C., from about 20° C. to about 55° C., from about 20° C. to about 50° C., from about 20° C. to about 45° C., from about 20° C. to about 40° C., from about 20° C. to about 35° C., from about 20° C. to about 30° C., from about 20° C. to about 25° C., from about 25° C. to about 60° C., from about 25° C. to about 55° C., from about 25° C. to about 50° C., from about 25° C. to about 45° C., from about 25° C. to about 40° C., from about 25° C. to about 35° C., from about 25° C. to about 30° C., from about 30° C. to about 60° C., from about 30° C. to about 55° C., from about 30° C. to about 50° C., from about 30° C. to about 45° C., from about 30° C. to about 40° C., from about 30° C. to about 35° C., from about 35° C. to about 60° C., from about 35° C. to about 55° C., from about 35° C. to about 50° C., from about 35° C. to about 45° C., from about 35° C. to about 40° C., from about 40° C. to about 60° C., from about 40° C. to about 55° C., from about 40° C. to about 50° C., from about 40° C. to about 45° C., from about 45° C. to about 60° C., from about 45° C. to about 55° C., from about 45° C. to about 50° C., from about 50° C. to about 60° C., from about 50° C. to about 55° C., or from about 55° C. to about 60° C. In some embodiments, NEAR may be performed above about 15° C., above about 20° C., above about 25° C., above about 30° C., above about 35° C., above about 40° C., above about 45° C., or above about 50° C. In some embodiments, an SDA reaction may be performed below about 60° C., below about 55° C., below about 50° C., below about 45° C., below about 40° C., below about 35° C., below about 30° C., below about 25° C., below about 20° C., or below about 15° C. In some embodiments, NEAR may be performed at about room temperature. In some embodiments, a nucleic acid sample may be heated prior to isothermal amplification. In some embodiments, the nucleic acid sample heated prior to isothermal amplification may comprise one or more primers. The nucleic acid sample may be heated to about 95° C. prior to isothermal amplification. The nucleic acid sample may be heated to a temperature sufficient to dissociate two strands of a double stranded nucleic acid sequence.
NEAR may amplify a target nucleic acid to detectable levels within about 30 seconds, 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. NEAR may amplify a target nucleic acid to detectable levels within about 1 hour, about 1.1 hours, about 1.2 hours, about 1.3 hours, about 1.4 hours, about 1.5 hours, about 1.6 hours, about 1.7 hours, about 1.8 hours, about 1.9 hours, about 2 hours, about 2.2 hours, about 2.4 hours, about 2.5 hours, about 2.6 hours, about 2.8 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours. NEAR may amplify a target nucleic acid to detectable levels within from about 0.1 hours to about 0.5 hours, from about 0.1 hours to about 1 hour, from about 0.1 hours to about 1.5 hours, from about 0.1 hours to about 2 hours, from about 0.1 hours to about 2.5 hours, from about 0.1 hours to about 3 hours, from about 0.1 hours to about 3.5 hours, from about 0.1 hours to about 4 hours, from about 0.1 hours to about 4.5 hours, from about 0.1 hours to about 5 hours, from about 0.5 hours to about 1 hour, from about 0.5 hours to about 1.5 hours, from about 0.5 hours to about 2 hours, from about 0.5 hours to about 2.5 hours, from about 0.5 hours to about 3 hours, from about 0.5 hours to about 3.5 hours, from about 0.5 hours to about 4 hours, from about 0.5 hours to about 4.5 hours, from about 0.5 hours to about 5 hours, from about 1 hour to about 1.5 hours, from about 1 hour to about 2 hours, from about 1 hour to about 2.5 hours, from about 1 hour to about 3 hours, from about 1 hour to about 3.5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 4.5 hours, from about 1 hour to about 5 hours, from about 1.5 hours to about 2 hours, from about 1.5 hours to about 2.5 hours, from about 1.5 hours to about 3 hours, from about 1.5 hours to about 3.5 hours, from about 1.5 hours to about 4 hours, from about 1.5 hours to about 4.5 hours, from about 1.5 hours to about 5 hours, from about 2 hours to about 2.5 hours, from about 2 hours to about 3 hours, from about 2 hours to about 3.5 hours, from about 2 hours to about 4 hours, from about 2 hours to about 4.5 hours, from about 2 hours to about 5 hours, from about 2.5 hours to about 3 hours, from about 2.5 hours to about 3.5 hours, from about 2.5 hours to about 4 hours, from about 2.5 hours to about 4.5 hours, from about 2.5 hours to about 5 hours, from about 3 hours to about 3.5 hours, from about 3 hours to about 4 hours, from about 3 hours to about 4.5 hours, from about 3 hours to about 5 hours, from about 3.5 hours to about 4 hours, from about 3.5 hours to about 4.5 hours, from about 3.5 hours to about 5 hours, from about 4 hours to about 4.5 hours, from about 4 hours to about 5 hours, or from about 4.5 hours to about 5 hours.
NEAR reaction components may comprise a polymerase, a nicking enzyme, dNTPs, and one or more nucleic acid primers. In some embodiments, the reaction may further comprise a reverse transcriptase as described herein. In some embodiments, the polymerase may be an exo-Klenow polymerase. The nicking enzyme may be capable of nicking a single strand of a double stranded nucleic acid sequence. In some embodiments, the nicking enzyme may be capable of nicking an unthiolated strand of a double stranded nucleic acid sequence comprising a thiolated strand and an unthiolated strand. In some embodiments, the nicking enzyme may be capable of nicking a single strand comprising an unthiolated region of a double stranded nucleic acid comprising at one or more thiolated regions and one or more unthiolated regions. In some embodiments, the nicking enzyme is a restriction enzyme capable of nicking a single strand of a double stranded nucleic acid sequence. In some embodiments, the nicking enzyme is a modified restriction enzyme. The nicking enzyme may be a strand-limited restriction enzyme. The restriction enzyme may be HincII. In some embodiments, the restriction enzyme may be AluI, BamHI, EcoP15I, EcoRI, EcoRII, EcoRV, HaeIII, HgaI, HindII, HindIII, HinFI, KpnI, NotI, PstI, PvuII, SacI, SalI, Sau3, ScaI, SmaI, SpeI, SphI, StuI, TaqI, or XbaI, or the like. The nicking enzyme may be Nt.BspQI, Nt.CvPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt. AlwI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt. BsmAI, Nb.Bpu10I, Nt. Bpu1101, Nb. Mva1269I, or I-Hmul, or the like. The one or more nucleic acid primers may comprise two primers. For example, the one or more nucleic acid primers may comprise a first primer (e.g., an S1 primer) and a second primer (e.g., an S2 primer). The target nucleic acid may be single stranded DNA or double stranded DNA. In some embodiments, a target nucleic acid comprising RNA may be reverse transcribed into DNA using a reverse transcriptase prior to NEAR amplification. A reverse transcription reaction may comprise primers, dNTPs, and a reverse transcriptase. In some embodiments, the reverse transcription reaction and the NEAR amplification reaction may be performed in the same reaction. A combined RT-NEAR reaction may comprise NEAR primers, reverse transcription primers, dNTPs, a reverse transcriptase, a polymerase, and dNTPs. In some embodiment, the NEAR primers may comprise the reverse transcription primers.
A DETECTR reaction to detect the target nucleic acid sequence may comprise a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease. The programmable nuclease when activated, as described elsewhere herein, exhibits sequence-independent cleavage of a reporter (e.g., a nucleic acid comprising a moiety that becomes detectable upon cleavage of the nucleic acid by the programmable nuclease). The programmable nuclease is activated upon the guide nucleic acid hybridizing to the target nucleic acid. A combined NEAR DETECTR reaction may comprise a polymerase, a restriction enzyme, dNTPs, one or more nucleic acid primers, a guide nucleic acid, a programmable nuclease, and a substrate nucleic acid. A combined RT-NEAR DETECTR reaction may comprise reverse transcription primers, a reverse transcriptase, a polymerase, a restriction enzyme, dNTPs, one or more nucleic acid primers, a guide nucleic acid, a programmable nuclease, and a substrate nucleic acid. In some embodiment, the primers may comprise the reverse transcription primers. NEAR and DETECTR can be carried out in the same sample volume. NEAR and DETECTR can be carried out concurrently in separate sample volumes or in the same sample volume. RT-NEAR and DETECTR can be carried out in the same sample volume. RT-NEAR and DETECTR can be carried out concurrently in separate sample volumes or in the same sample volume. A NEAR reaction may be multiplexed to amplify a plurality of target nucleic acid sequences in a single reaction.
A number of NEAR primers and NEAR primer design methods are consistent with the methods compositions, reagents, enzymes, and kits disclosed herein. NEAR may comprise a set of primers. In some embodiments, NEAR may be an RT-NEAR reaction, a NEAR DETECTR reaction, or an RT-NEAR DETECTR reaction. The set of NEAR primers may comprise two primers, a first primer and a second primer. In some embodiments, a first primer may comprise a sequence of the first region at the 3′ end of the first primer. The sequence of the first region may be from about 16 nucleic acids to about 25 nucleic acids long, or about 20 nucleic acids long. The 3′ end of the first primer may hybridize to the first complementary region of the target. The first complementary region may be 3′ of the target nucleic acid. The first complementary region may be 3′ of a sequence reverse complementary to the target nucleic acid. The first primer may further comprise a cut site 5′ of the sequence of the first region that may be recognized and cleaved by a nicking enzyme. The 3′ end of the first primer may further comprise a recognition site for a nicking enzyme. In some embodiments, the 3′ end of the first primer may further comprise a nicking enzyme stabilization region. The sequence of the second region may be from about 30 nucleic acids to about 38 nucleic acids long. The 3′ end of the second primer may hybridize to the second complementary region. The second complementary region may be 3′ of a sequence reverse complementary to the target nucleic acid. The second complementary region may be 3′ of the target nucleic acid. The second complementary region may be 3′ of a sequence reverse complementary to the target nucleic acid. The second primer may further comprise a cut site 5′ of the sequence of the second region that may be recognized and cleaved by a nicking enzyme. The 3′ end of the second primer may further comprise a recognition site for a nicking enzyme. In some embodiments, the 3′ end of the second primer may further comprise a nicking enzyme stabilization region.
The NEAR primers are designed depending on the site of the optimal guide RNA placement, which may or may not be determined by an available PAM sequence. When performing a NEAR-DETECTR reaction, single-stranded DNA is produced by the designed primers. Because the DETECTR reaction will detection single stranded DNA species, the amplification reaction can be biased to produce more of the particular strand than another. This can be done through changing of the ratio of the forward and reverse primer concentrations. In some embodiments, the concentration of forward primer can be 5 times, 4 times, 3 times, 2 times, or equal to the concentration of reverse primer. In some embodiments, the concentration of reverse primer can be 5 times, 4 times, 3 times, 2 times, or equal to the concentration of forward primer.
In some embodiments, the first region, the second region, or both may be about 8 nucleic acids, about 10 nucleic acids, about 12 nucleic acids, about 14 nucleic acids, about 16 nucleic acids, about 18 nucleic acids, about 20 nucleic acids, about 22 nucleic acids, about 24 nucleic acids, about 26 nucleic acids, about 28 nucleic acids, about 30 nucleic acids, about 32 nucleic acids, about 34 nucleic acids, about 36 nucleic acids, about 38 nucleic acids, about 40 nucleic acids, about 42 nucleic acids, about 44 nucleic acids, about 46 nucleic acids, about 48 nucleic acids, or about 50 nucleic acids long.
In some embodiments, the first region, the second region, or both may be from about 8 to about 12, from about 8 to about 16, from about 8 to about 20, from about 8 to about 24, from about 8 to about 28, from about 8 to about 30, from about 8 to about 32, from about 8 to about 34, from about 8 to about 36, from about 8 to about 38, from about 8 to about 40, from about 8 to about 42, from about 8 to about 44, from about 8 to about 48, from about 8 to about 50, from about 12 to about 16, from about 12 to about 20, from about 12 to about 24, from about 12 to about 28, from about 12 to about 30, from about 12 to about 32, from about 12 to about 34, from about 12 to about 36, from about 12 to about 38, from about 12 to about 40, from about 12 to about 42, from about 12 to about 44, from about 12 to about 48, from about 12 to about 50, from about 16 to about 20, from about 16 to about 24, from about 16 to about 28, from about 16 to about 30, from about 16 to about 32, from about 16 to about 34, from about 16 to about 36, from about 16 to about 38, from about 16 to about 40, from about 16 to about 42, from about 16 to about 44, from about 16 to about 48, from about 16 to about 50, from about 20 to about 24, from about 20 to about 28, from about 20 to about 30, from about 20 to about 32, from about 20 to about 34, from about 20 to about 36, from about 20 to about 38, from about 20 to about 40, from about 20 to about 42, from about 20 to about 44, from about 20 to about 48, from about 20 to about 50, from about 24 to about 28, from about 24 to about 30, from about 24 to about 32, from about 24 to about 34, from about 24 to about 36, from about 24 to about 38, from about 24 to about 40, from about 24 to about 42, from about 24 to about 44, from about 24 to about 48, from about 24 to about 50, from about 28 to about 30, from about 28 to about 32, from about 28 to about 34, from about 28 to about 36, from about 28 to about 38, from about 28 to about 40, from about 28 to about 42, from about 28 to about 44, from about 28 to about 48, from about 28 to about 50, from about 30 to about 32, from about 30 to about 34, from about 30 to about 36, from about 30 to about 38, from about 30 to about 40, from about 30 to about 42, from about 30 to about 44, from about 30 to about 48, from about 30 to about 50, from about 32 to about 34, from about 32 to about 36, from about 32 to about 38, from about 32 to about 40, from about 32 to about 42, from about 32 to about 44, from about 32 to about 48, from about 32 to about 50, from about 34 to about 36, from about 34 to about 38, from about 34 to about 40, from about 34 to about 42, from about 34 to about 44, from about 34 to about 48, from about 34 to about 50, from about 36 to about 38, from about 36 to about 40, from about 36 to about 42, from about 36 to about 44, from about 36 to about 48, from about 36 to about 50, from about 38 to about 40, from about 38 to about 42, from about 38 to about 44, from about 38 to about 48, from about 38 to about 50, from about 40 to about 42, from about 40 to about 44, from about 40 to about 48, from about 40 to about 50, from about 42 to about 44, from about 42 to about 48, from about 42 to about 50, from about 44 to about 48, from about 44 to about 50, from about 48 to about 50 nucleic acids long.
In some embodiments, the first region, the second region, or both may comprise a GC content of about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In some embodiments, the first region, the second region, or both may comprise a GC content of from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 15%, from about 1% to about 20%, from about 1% to about 25%, from about 1% to about 30%, from about 1% to about 40%, from about 1% to about 50%, from about 1% to about 60%, from about 1% to about 70%, from about 1% to about 80%, from about 1% to about 90%, from about 1% to about 95%, from about 1% to about 99%, from about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 25%, from about 5% to about 30%, from about 5% to about 40%, from about 5% to about 50%, from about 5% to about 60%, from about 5% to about 70%, from about 5% to about 80%, from about 5% to about 90%, from about 5% to about 95%, from about 5% to about 99%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 30%, from about 10% to about 40%, from about 10% to about 50%, from about 10% to about 60%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, from about 10% to about 95%, from about 10% to about 99%, from about 15% to about 20%, from about 15% to about 25%, from about 15% to about 30%, from about 15% to about 40%, from about 15% to about 50%, from about 15% to about 60%, from about 15% to about 70%, from about 15% to about 80%, from about 15% to about 90%, from about 15% to about 95%, from about 15% to about 99%, from about 20% to about 25%, from about 20% to about 30%, from about 20% to about 40%, from about 20% to about 50%, from about 20% to about 60%, from about 20% to about 70%, from about 20% to about 80%, from about 20% to about 90%, from about 20% to about 95%, from about 20% to about 99%, from about 25% to about 30%, from about 25% to about 40%, from about 25% to about 50%, from about 25% to about 60%, from about 25% to about 70%, from about 25% to about 80%, from about 25% to about 90%, from about 25% to about 95%, from about 25% to about 99%, from about 30% to about 40%, from about 30% to about 50%, from about 30% to about 60%, from about 30% to about 70%, from about 30% to about 80%, from about 30% to about 90%, from about 30% to about 95%, from about 30% to about 99%, from about 40% to about 50%, from about 40% to about 60%, from about 40% to about 70%, from about 40% to about 80%, from about 40% to about 90%, from about 40% to about 95%, from about 40% to about 99%, from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, from about 50% to about 95%, from about 50% to about 99%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, from about 60% to about 95%, from about 60% to about 99%, from about 70% to about 80%, from about 70% to about 90%, from about 70% to about 95%, from about 70% to about 99%, from about 80% to about 90%, from about 80% to about 95%, from about 80% to about 99%, from about 90% to about 95%, from about 90% to about 99%, or from about 95% to about 99%.
In some embodiments, the first region, the second region, or both may have a melting temperature of about 38° C., about 40° C., about 42° C., about 44° C., about 46° C., about 48° C., about 50° C., about 52° C., about 54° C., about 56° C., about 58° C., about 60° C., about 62° C., about 64° C., about 66° C., about 68° C., about 70° C., about 72° C., about 74° C., about 76° C., about 78° C., about 80° C., about 82° C., about 84° C., about 86° C., about 88° C., about 90° C., or about 92° C. In some embodiments, the first region, the second region, or both may have a melting temperature of from about 35° C. to about 40° C., from about 35° C. to about 45° C., from about 35° C. to about 50° C., from about 35° C. to about 55° C., from about 35° C. to about 60° C., from about 35° C. to about 65° C., from about 35° C. to about 70° C., from about 35° C. to about 75° C., from about 35° C. to about 80° C., from about 35° C. to about 85° C., from about 35° C. to about 90° C., from about 35° C. to about 95° C., from about 40° C. to about 45° C., from about 40° C. to about 50° C., from about 40° C. to about 55° C., from about 40° C. to about 60° C., from about 40° C. to about 65° C., from about 40° C. to about 70° C., from about 40° C. to about 75° C., from about 40° C. to about 80° C., from about 40° C. to about 85° C., from about 40° C. to about 90° C., from about 40° C. to about 95° C., from about 45° C. to about 50° C., from about 45° C. to about 55° C., from about 45° C. to about 60° C., from about 45° C. to about 65° C., from about 45° C. to about 70° C., from about 45° C. to about 75° C., from about 45° C. to about 80° C., from about 45° C. to about 85° C., from about 45° C. to about 90° C., from about 45° C. to about 95° C., from about 50° C. to about 55° C., from about 50° C. to about 60° C., from about 50° C. to about 65° C., from about 50° C. to about 70° C., from about 50° C. to about 75° C., from about 50° C. to about 80° C., from about 50° C. to about 85° C., from about 50° C. to about 90° C., from about 50° C. to about 95° C., from about 55° C. to about 60° C., from about 55° C. to about 65° C., from about 55° C. to about 70° C., from about 55° C. to about 75° C., from about 55° C. to about 80° C., from about 55° C. to about 85° C., from about 55° C. to about 90° C., from about 55° C. to about 95° C., from about 60° C. to about 65° C., from about 60° C. to about 70° C., from about 60° C. to about 75° C., from about 60° C. to about 80° C., from about 60° C. to about 85° C., from about 60° C. to about 90° C., from about 60° C. to about 95° C., from about 65° C. to about 70° C., from about 65° C. to about 75° C., from about 65° C. to about 80° C., from about 65° C. to about 85° C., from about 65° C. to about 90° C., from about 65° C. to about 95° C., from about 70° C. to about 75° C., from about 70° C. to about 80° C., from about 70° C. to about 85° C., from about 70° C. to about 90° C., from about 70° C. to about 95° C., from about 75° C. to about 80° C., from about 75° C. to about 85° C., from about 75° C. to about 90° C., from about 75° C. to about 95° C., from about 80° C. to about 85° C., from about 80° C. to about 90° C., from about 80° C. to about 95° C., from about 85° C. to about 90° C., from about 85° C. to about 95° C., or from about 90° C. to about 95° C.
A set of NEAR primers may be designed for use in combination with a DETECTR reaction. The amplified nucleic acid sequence may comprise a sequence that hybridizes to a guide RNA. The amplified nucleic acid sequence may comprise a target nucleic acid. The guide RNA may hybridize to the target nucleic acid. The amplified nucleic acid sequence may comprise corresponding to a guide RNA. The amplified nucleic acid sequence may comprise a sequence reverse complementary to the target nucleic acid. All or part of the guide RNA may be reverse complementary to all or part of the target nucleic acid. The amplified nucleic acid sequence may comprise a protospacer adjacent motif (PAM) positioned next to or near the target sequence. The PAM may be 3′ of the target nucleic acid. In some embodiments, a portion of a sequence that hybridizes the guide RNA may be located between the first region and the second complementary region. The portion of a sequence that hybridizes the guide RNA located between the first region and the second complementary region may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more of the sequence that hybridizes guide RNA. In some embodiments, the 5′ end of the sequence that hybridizes the guide RNA is 3′ of the 3′ end of the first region and 3′ end of the sequence that hybridizes the guide RNA is 5′ of the 5′ end of the second complementary region. In some embodiments, a portion of a sequence that hybridizes the guide RNA may be located between the second region and the first complementary region. The portion of a sequence that hybridizes the guide RNA located between the second region and the first complementary region may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more of the sequence that hybridizes the guide RNA. In some embodiments, the 5′ end of the sequence that hybridizes the guide RNA is 3′ of the 3′ end of the second region and 3′ end of the sequence that hybridizes the guide RNA is 5′ of the 5′ end of the first complementary region.
In some embodiments, a sequence that hybridizes the guide RNA may overlap the first region, the first complementary region, the second region, or the second complementary region by no more than no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 35%, no more than about 40%, no more than about 45%, no more than about 50%, no more than about 55%, no more than about 60%, no more than about 65%, no more than about 70%, no more than about 75%, no more than about 80%, no more than about 85%, no more than about 90%, or no more than about 95%. In some embodiments, the sequence that hybridizes the guide RNA does not overlap the first region, the first complementary region, the second region, or the second complementary region. In some embodiments, the guide RNA does not hybridize to the first primer or the second primer.
In some embodiments, a NEAR primer set may be designed using a commercially available primer design software. A NEAR primer set may be designed for use in combination with a DETECR reaction, a reverse transcription reaction, or both. One or more methods of designing a set of NEAR primers may be readily apparent to one skilled in the art and may be employed in any of the compositions, kits and methods described herein.
A DETECTR reaction may be used to detect the presence of a specific target gene in the same. The DETECTR reaction may produce a detectable signal, as described elsewhere herein, in the presence of a target nucleic acid sequence comprising a target gene. The DETECTR reaction may not produce a signal in the absence of the target nucleic acid or in the presence of a nucleic acid sequence that does not comprise the specific SNP allele or comprises a different SNP allele. In some embodiments, a DETECTR reaction may comprise a guide RNA reverse complementary to a portion of a target nucleic acid sequence comprising a specific SNP allele. The guide RNA and the target nucleic acid comprising the specific SNP allele may bind to and activate a programmable nuclease, thereby producing a detectable signal as described elsewhere herein. The guide RNA and a nucleic acid sequence that does not comprise the specific SNP allele may not bind to or activate the programmable nuclease and may not produce a detectable signal. In some embodiments, a target nucleic acid sequence that may or may not comprise a specific SNP allele may be amplified using, for example, a LAMP amplification reaction, an RPA amplification reaction, an SDA amplification reaction, a NEAR amplification reaction, or any other amplification method. In some embodiments, the amplification reaction may be combined with a reverse transcription reaction, a DETECTR reaction, or both. For example, the amplification reaction may be an RT-NEAR reaction, a NEAR DETECTR reaction, or an RT-NEAR DETECTR reaction. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.
A DETECTR reaction, as described elsewhere herein, may produce a detectable signal specifically in the presence of a target nucleic acid sequence comprising a target gene. In addition to the DETECTR reaction, the target nucleic acid having the target gene may be concurrently, sequentially, concurrently together in a sample, or sequentially together in a sample be carried out alongside NEAR or RT-NEAR. For example, the reactions can comprise NEAR and DETECTR reactions, or RT-NEAR and DETECTR reactions. Performing a DETECTR reaction in combination with a NEAR reaction may result in an increased detectable signal as compared to the DETECTR reaction in the absence of the NEAR reaction. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.
In some embodiments, the detectable signal produced in the DETECTR reaction may be higher in the presence of a target nucleic acid comprising target nucleic acid than in the presence of a nucleic acid that does not comprise the target nucleic acid. In some embodiments, the DETECTR reaction may produce a detectable signal that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at last 400-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold greater in the presence of a target nucleic acid comprising a target nucleic acid than in the presence of a nucleic acid that does not comprise the target nucleic acid. In some embodiments, the DETECTR reaction may produce a detectable signal that is from 1-fold to 2-fold, from 2-fold to 3-fold, from 3-fold to 4-fold, from 4-fold to 5-fold, from 5-fold to 10-fold, from 10-fold to 20-fold, from 20-fold to 30-fold, from 30-fold to 40-fold, from 40-fold to 50-fold, from 50-fold to 100-fold, from 100-fold to 500-fold, from 500-fold to 1000-fold, from 1000-fold to 10,000-fold, from 10,000-fold to 100,000-fold, or from 100,000-fold to 1,000,000-fold greater in the presence of a target nucleic acid comprising a specific SNP allele than in the presence of a nucleic acid that does not comprise the specific SNP allele. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.
A DETECTR reaction may be used to detect the presence of a target nucleic acid associated with a disease or a condition in a nucleic acid sample. The DETECTR reaction may reach signal saturation within about 30 seconds, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 75 minutes, about 80 minutes, or about 85 minutes and be used to detect the presence of a target gene associated with an increased likelihood of developing a disease or a condition in a nucleic acid sample. The DETECTR reaction may be used to detect the presence of a target gene associated with a phenotype in a nucleic acid sample. For example, a DETECTR reaction may be used to detect target gene associated with a disease such as phenylketonuria (PKU), cystic fibrosis, sickle-cell anemia, albinism, Huntington's disease, myotonic dystrophy type 1, hypercholesterolemia, neurofibromatosis, polycystic kidney disease, hemophilia, muscular dystrophy, hypophosphatemic rickets, Rett's syndrome, or spermatogenic failure. A DETECTR reaction may be used to detect a SNP allele associated with an increased risk of cancer, for example bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, gallbladder cancer, stomach cancer, leukemia, liver cancer, lung cancer, oral cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, thyroid cancer, neuroblastoma, or lymphoma. A DETECTR reaction may be used to detect a SNP allele associated with an increased risk of a disease, for example Alzheimer's disease, Parkinson's disease, amyloidosis, heterochromatosis, celiac disease, macular degeneration, or hypercholesterolemia. A DETECTR reaction may be used to detect a SNP allele associated with a phenotype, for example, eye color, hair color, height, skin color, race, alcohol flush reaction, caffeine consumption, deep sleep, genetic weight, lactose intolerance, muscle composition, saturated fat and weight, or sleep movement. A DETECTR reaction may also be used to detect the presence of a pathological organism. In some embodiments, the pathological organism is a prokaryote, eukaryote, or a protozoa. In some embodiments, the pathological organism is a virus, an opportunistic pathogen, a parasite, a bacterium, or any combination thereof. In some embodiments, the pathological organism is SARS-CoV-2 or Streptococcus pyogenes.
The terms “sample interface”, “sample input”, “input port”, “input”, “port” as used herein, generally refers to a portion of a device that is configured to receive a sample.
The terms “heating region”, “heated region”, “heat chambers”, “heat volumes”, “heat zones”, “heat surfaces”, “heat areas”, and the like, as used herein, generally refers to a portion of a device that is in thermal communication with a heating unit.
The terms “heater”, “heating unit”, “heating element”, “heat source”, and the like, as used herein, generally refers to an element that is configured to produce heat and is in thermal communication with a portion of a device.
The term “reagent mix”, “reagent master mix”, “reagents”, and the like, as used herein, generally refers to a formulation comprising one or more chemicals that partake in a reaction that the reagent mix is formulated for.
The term “non-cycled temperature profile,” as used herein, generally refers to a temperature profile that is cyclical or sinusoidal in that the temperature profile has an initial temperature, a target temperature, and a final temperature.
The term “capture probe”, “capture molecule”, and the like, as used herein, generally refers to a molecule that selectively binds to a target nucleic acid and only nonspecifically binds to other nucleic acids that can be washed away.
The term “collection tube,” as used herein, generally refers to a compartment that is used to collect a sample and deliver the sample to the sample interface of a device. In some embodiments, the collection tube may be portable. In some embodiments, the collection tube is a syringe.
As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/−0.1% of the stated value (or range of values), +/−1% of the stated value (or range of values), +/−2% of the stated value (or range of values), +/−5% of the stated value (or range of values), or +/−10% of the stated value (or range of values). Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points.
As used herein, the terms “thermostable” and “thermostability” refer to the stability of a composition disclosed herein at one or more temperatures, such as an elevated operating temperature for a given reaction. Stability may be assessed by the ability of the composition to perform an activity, e.g., cleaving a target nucleic acid or reporter. Improving thermostability means improving the quantity or quality of the activity at one or more temperatures.
As used herein, the terms “percent identity,” “% identity,” and “% identical” refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X % identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X % of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y in an alignment between the two. Generally, computer programs may be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar.; 4 (1): 11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. 1988 Apr; 85 (8): 2444-8; Pearson, Methods Enzymol. 1990; 183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep. 1; 25 (17): 3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan. 11; 12 (1 Pt 1): 387-95). For the purposes of calculating identity to the sequence, extensions, such as tags, are not included.
As used herein, a “one-pot” reaction refers to a reaction in which more than one reaction occurs in a single volume alongside a programmable nuclease-based detection (e.g., DETECTR) assay. For example, in a one-pot assay, sample preparation, reverse transcription, amplification, in vitro transcription, or any combination thereof, and programmable nuclease-based detection (e.g., DETECTR) assays are carried out in a single volume. In some embodiments, amplification and detection are carried out within a same volume or region of a device (e.g., within a detection region). Readout of the detection (e.g., DETECTR) assay may occur in the single volume or in a second volume. For example, the product of the one-pot DETECTR reaction (e.g., a cleaved detection moiety comprising an enzyme) may be transferred to another volume (e.g., a volume comprising an enzyme substrate) for signal generation and indirect detection of reporter cleavage by a sensor or detector (or by eye in the case of a colorimetric signal).
As used herein, “HotPot” refers to a one-pot reaction in which both amplification (e.g., RT-LAMP) and detection (e.g., DETECTR) reactions occur simultaneously. In many embodiments, a HotPot reaction may utilize a thermostable programmable nuclease which exhibits trans cleavage at elevated temperatures (e.g., greater than 37C).
The terms, “amplification” and “amplifying,” as used herein, refer to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof.
The term, “complementary,” as used herein with reference to a nucleic acid refers to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T/U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5′- to 3′-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3′- to its 5′-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5′- to its 3′-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.
The term, “cleavage assay,” as used herein refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid. In some cases, the cleavage activity may be cis-cleavage activity. In some cases, the cleavage activity may be trans-cleavage activity.
Assays which leverage the transcollateral cleavage properties of programmable nuclease enzymes (e.g., CRISPR-Cas enzymes) are often referred to herein as DNA endonuclease targeted CRISPR trans reporter (DETECTR) reactions. As used herein, detection of reporter cleavage (directly or indirectly) to determine the presence of a target nucleic acid sequence may be referred to as “DETECTR”.
The term, “detectable signal,” as used herein refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.
The term, “detecting a nucleic acid” and its grammatical equivalents, as used herein refers to detecting the presence or absence of the target nucleic acid in a sample that potentially contains the nucleic acid being detected.
The term, “effector protein,” as used herein refers to a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. In some embodiments, the complex comprises multiple effector proteins. In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some embodiments, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid. A non-limiting example of modifying a target nucleic acid is cleaving (hydrolysis) of a phosphodiester bond. Additional examples of modifying target nucleic acids are described herein and throughout. In some embodiments, the term, “effector protein” refers to a protein that is capable of modifying a nucleic acid molecule (e.g., by cleavage, deamination, recombination). Modifying the nucleic acid may modulate the expression of the nucleic acid molecule (e.g., increasing or decreasing the expression of a nucleic acid molecule). The effector protein may be a Cas protein (i.e., an effector protein of a CRISPR-Cas system).
The term, “guide nucleic acid,” as used herein refers to a nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of being non-covalently bound by an effector protein. The first sequence may be referred to herein as a spacer sequence. The second sequence may be referred to herein as a repeat sequence. In some embodiments, the first sequence is located 5′ of the second nucleotide sequence. In some embodiments, the first sequence is located 3′ of the second nucleotide sequence.
The terms, “non-naturally occurring” and “engineered,” as used herein are used interchangeably and indicate the involvement of human intervention. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and/or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non-limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by human intervention.
The term, “protospacer adjacent motif (PAM),” as used herein refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. A PAM sequence may be required for a complex having an effector protein and a guide nucleic acid to hybridize to and modify the target nucleic acid. However, a given effector protein may not require a PAM sequence being present in a target nucleic acid for the effector protein to modify the target nucleic acid.
The terms, “reporter” and “reporter nucleic acid,” are used interchangeably herein to refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein.
The term, “sample,” as used herein generally refers to something comprising a target nucleic acid. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of non-limiting example, samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.
The term, “target nucleic acid,” as used herein refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA).
The term, “target sequence,” as used herein when used in reference to a target nucleic acid refers to a sequence of nucleotides that hybridizes to a portion (preferably an equal length portion) of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
The present disclosure provides the following illustrative embodiments.
Embodiment 1. A device for detecting a target nucleic acid, the device comprising:
Embodiment 2. A method for detecting a plurality of target nucleic acids, the method comprising:
Embodiment 3. A device for detecting a target nucleic acid, the device comprising:
Embodiment 4. A device for detecting a target nucleic acid, comprising:
Embodiment 5. A device for detecting a target nucleic acid, the device comprising:
Embodiment 6. A device for detecting a target nucleic acid, comprising:
Embodiment 7. A device for detecting a target nucleic acid, comprising:
Embodiment 8. A device for detecting a target nucleic acid, comprising:
Embodiment 9. The device of Embodiment 8, wherein the programmable nuclease-enzyme fusion protein comprises Cas12-HRP.
Embodiment 10. The device of Embodiment 9, wherein the tether of each reporter of the plurality of reporters comprises ssDNA.
Embodiment 11. The device of Embodiment 9, wherein the tether of each reporter of the plurality of reporters comprises RNA.
Embodiment 12. The device of Embodiment 11, wherein the tether of each reporter of the plurality of reporters further comprises DNA.
Embodiment 13. The device of Embodiment 8, wherein the programmable nuclease-enzyme fusion protein comprises Cas12-HRP.
Embodiment 14. The device of Embodiment 13, wherein the tether of each reporter of the plurality of reporters comprises RNA.
Embodiment 15. The device of any one of Embodiments 8-14, wherein the enzyme substrate is HRP substrate.
Embodiment 16. The device of Embodiment 8, wherein the surface is configured to immobilize a nucleic acid.
Embodiment 17. The device of Embodiment 8, wherein the surface comprises streptavidin, biotin, an amine group, a carboxyl group, an epoxy group, an NHS group, a malemide group, or a thiol group.
Embodiment 18. The device of Embodiment 8, wherein the surface is a hydrogel surface.
Embodiment 19. The device of Embodiment 18, wherein the surface comprises an acrydite modification.
Embodiment 20. A device for detecting a target nucleic acid, comprising:
Embodiment 21. The device of Embodiment 20, wherein the programmable nuclease-split enzyme fusion protein comprises HRP-L.
Embodiment 22. The device of Embodiment 21, further comprising a programmable nuclease-split enzyme fusion protein comprising HRP-S.
Embodiment 23. The device of Embodiment 22, wherein the device is configured to produce a signal upon binding of the programmable nuclease-split enzyme fusion protein comprising HRP-L and binding of the programmable nuclease-split enzyme fusion protein comprising HRP-S to the target nucleic acid.
Embodiment 24. A device for detecting a target nucleic acid, comprising:
Embodiment 25. The device of Embodiment 24, wherein each enzyme of the two or more enzymes comprises HRP.
Embodiment 26. The device of Embodiment 24, wherein the reporter comprises a poly-HRP comprising a streptavidin comprising the two or more enzymes.
Embodiment 27. The device of Embodiment 24, wherein the tether contacts the surface via a functionality optionally comprising a biotin, an acrydite, or an amine.
Embodiment 28. The device of Embodiment 24, wherein the surface of the first reaction chamber is functionalized with a hydrogel, carboxyl group, or N-Hydroxysuccinimide (NHS).
Embodiment 29. The device of Embodiment 24, wherein the tether comprises a segment of cleavable DNA, cleavable RNA, or a combination thereof.
Embodiment 30. The device of Embodiment 24 or 25, wherein the enzyme substrate comprises 3,3′,5,5′-tetramethylbenzidine (TMB).
Embodiment 31. The device of the Embodiment 24, wherein the surface of the first reaction chamber comprises the inner wall of the first reaction chamber.
Embodiment 32. The device of Embodiment 24, wherein the surface of the first reaction chamber comprises a surface of a bead, wherein the bead is contained within the first reaction chamber.
Embodiment 33. The device of Embodiment 32, wherein the bead is a magnetic bead.
Embodiment 34. A composition comprising a plurality of reporters, wherein (a) each reporter of the plurality of reporters comprises one or more enzymes conjugated to a linker; (b) the linker comprises one or more nucleic acid sections; and (c) the linker (i) comprises a functionality for immobilization to a support, or (ii) is conjugated to the support.
Embodiment 35. The composition of Embodiment 34, wherein the one or more enzymes is a single enzyme.
Embodiment 36. The composition of Embodiment 35, wherein the enzyme is a horseradish peroxidase (HRP).
Embodiment 37. The composition of Embodiment 34, wherein the one or more enzymes comprises a poly-HRP comprising a plurality of HRP enzymes.
Embodiment 38. The composition of Embodiment 37, wherein the plurality of HRP enzymes of the poly-HRP are complexed with streptavidin, and the linker comprises a biotin for conjugation with the poly-HRP.
Embodiment 39. The composition of any one of Embodiments 34-38, wherein the one or more nucleic acid sections comprise one or more sections of DNA, one or more sections of RNA, or a combination thereof.
Embodiment 40. The composition of Embodiment 39, wherein the one or more nucleic acid sections consist of DNA.
Embodiment 41. The composition of Embodiment 39, wherein the one or more nucleic acid sections consist of RNA.
Embodiment 42. The composition of Embodiment 39, wherein the one or more nucleic acid sections comprise one or more sections of DNA and one or more sections of RNA.
Embodiment 43. The composition of any one of Embodiments 39-42, wherein the one or more nucleic acid sections are single-stranded.
Embodiment 44. The composition of any one of Embodiments 39-43, wherein the one or more nucleic acid sections have a combined length of at least 20, 30, 40, or 50 nucleotides.
Embodiment 45. The composition of any one of Embodiments 39-43, wherein at least one of the one or more nucleic acid sections is a substrate for cleavage by a programmable nuclease.
Embodiment 46. The composition of Embodiment 39, wherein the linker comprises (a) a first portion comprising a non-naturally occurring guide nucleic acid, and (b) a second portion comprising a nucleic acid section that is a substrate for cleavage by a programmable nuclease; and further wherein the one or more enzymes are conjugated to the linker via the second portion.
Embodiment 47. The composition of Embodiment 46, wherein the one or more enzymes comprise HRP.
Embodiment 48. The composition of any one of Embodiments 34-47, wherein the linker further comprises one or more hydrocarbon chains.
Embodiment 49. The composition of Embodiment 48, wherein the one or more hydrocarbon chains comprises a hydrocarbon chain between the one or more nucleic acid sections and the support.
Embodiment 50. The composition of Embodiment 48 or 49, wherein the one or more hydrocarbon chains comprises a hydrocarbon chain between the one or more nucleic acid sections and the one or more enzymes.
Embodiment 51. The composition of any one of Embodiments 34-50, wherein the support is a bead, a hydrogel, or the surface of a container.
Embodiment 52. The composition of any one of Embodiments 34-51, wherein the linker comprises the functionality for immobilization to the support.
Embodiment 53. The composition of any one of Embodiments 34-51, wherein the linker is conjugated to the support.
Embodiment 54. The composition of any one of Embodiments 34-53, further comprising a programmable nuclease.
Embodiment 55. A system comprising a reaction chamber, wherein the reaction chamber comprises the composition of any one of Embodiments 34-54.
Embodiment 56. A method for detecting a target nucleic acid in a sample, the method comprising
Embodiment 57. A method of detecting a target nucleic acid in a sample, the method comprising:
Embodiment 58. The method of Embodiment 56 or 57, further comprising concentrating the one or more enzymes prior to contacting the one or more enzymes with the substrate, wherein the concentrating comprises separating the one or more enzymes from at least a portion of the separated reaction fluid.
Embodiment 59. The method of Embodiment 58, wherein concentrating comprises capture of the enzyme on a support.
Embodiment 60. The method of Embodiment 59, wherein contacting the separated reaction fluid with the substrate of the one or more enzymes comprises contacting the enzyme on the support with the substrate 2, 3, 4, 5, or more times.
Embodiment 61. A composition for detecting a target nucleic acid, the composition comprising a programmable nuclease, a guide nucleic acid, a first reporter, an enzyme, and a second reporter, wherein:
Embodiment 62. The composition of Embodiment 61, wherein the enzyme is an endonuclease and the second reporter comprises a polynucleotide substrate of the enzyme.
Embodiment 63. The composition of Embodiment 62, wherein the endonuclease is a NucC endonuclease.
Embodiment 64. The composition of Embodiment 63, wherein the first nucleic acid section comprises adenosine residues.
Embodiment 65. The composition of Embodiment 64, wherein the adenosine residues comprise cyclic adenylate (cA3).
Embodiment 66. The composition of any one of Embodiments 61-65, wherein the second nucleic acid section comprises RNA residues, optionally wherein the RNA residues comprise a plurality of uracil residues.
Embodiment 67. The composition of any one of Embodiments 61-65, wherein the second nucleic acid section comprises DNA residues, optionally wherein the DNA residues comprise a plurality of thymine residues.
Embodiment 68. The composition of any one of Embodiments 61-67, wherein (a) the second reporter comprises a fluorescent label and a quencher, and (b) cleavage of the second reporter by the activated enzyme is effective to separate the fluorescent label from the quencher.
Embodiment 69. A method of detecting a target nucleic acid in a sample, the method comprising:
Embodiment 70. The method of Embodiment 69, wherein (a) the second reporter comprises a polynucleotide substrate of the enzyme, and (b) the enzyme is a NucC.
Embodiment 71. The method of Embodiment 70, wherein step (d) is performed at a temperature of at least 40° C.
Embodiment 72. A composition for detecting a target nucleic acid in a reaction chamber, the composition comprising a programmable nuclease, a guide nucleic acid, a forward primer, a reverse primer, a polymerase, a nicking endonuclease, and a reporter, wherein:
Embodiment 73. The composition of Embodiment 72, wherein (a) the sequence of the target nucleic acid to which the 3′ portion of the first primer is configured to bind defines a first sequence of the target nucleic acid; (b) the sequence of the 3′ portion of the reverse primer defines a second sequence of the target nucleic acid; and (c) the first sequence and second sequence are separated by about 5 to about 10 nucleotides along the target nucleic acid.
Embodiment 74. The composition of Embodiment 72 or 73, wherein the 3′ portions of the forward primer and reverse primer are about 16 to about 20 nucleotides in length.
Embodiment 75. The composition of any one of Embodiments 72-74, wherein overlap between the 3′ portion of the reverse primer and the sequence to which the guide nucleic acid is configured to bind overlap by 1 to 5 nucleotides, 2 to 5 nucleotides, or 3 nucleotides.
Embodiment 76. The composition of any one of Embodiments 72-75, wherein the first hairpin and/or the second hairpin are 10 to 20 nucleotides in length, 16 to 20 nucleotides in length, or 16 nucleotides in length.
Embodiment 77. The composition of any one of Embodiments 72-76, wherein the programmable nuclease is a Cas protein, optionally wherein the Cas protein is a Cas12 protein or a Cas14 protein.
Embodiment 78. A method of detecting a target nucleic acid in a sample, the method comprising:
Embodiment 79. The method of Embodiment 78, wherein steps (b) through (d) are performed at about the same temperature.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
DETECTR has previously been demonstrated to be a powerful technology for detection of pathogens such as SARS-CoV-2 (Broughton et al., 2020). Electrochemical detection has been demonstrated to show a lower limit of detection than fluorescence-based assays by roughly two orders of magnitude (Lou et al., 2015). In this example, an electrochemical probe is incorporated into the DETECTR assay.
The following electrochemical probe: 5′-2XXTTATTXX-3′; Where 2=5′ 6-FAM; X=ferrocene dT and 3=3′ Biotin TEG was used. Additionally, both cyclic voltammetry and square wave voltammetry with the probe, using a DropSens μSTAT. ECL instrument and DropSens screen-printed carbon electrodes were used. A difference in signal between the initial and final timepoints of the DETECTR reaction was observed. A detection of 50 fM. HERC2 target, which is much lower than has been observed with the fluorescence assay was achieved.
The following electrochemical probe: 5′-2XXTTATTXX-3′; where 2=5′ 6-FAM; X=ferrocene dT; and 3=3′ Biotin TEG was used with a DNA sequence, part of the genome of SARS-CoV-2. Square wave voltammetry measurements were performed using a DropSens μSTAT ECL instrument and DropSens screen-printed carbon electrodes. A difference in signal between the initial and final timepoints of the DETECTR reaction, using 500 fM of target, was detected.
Data Analysis was carried out as follows. The first 2 scans were discarded because they typically have anomalous signals from debris on the electrode that is cleared by voltage application. The remaining 3 scans are averaged for each sample, and the standard deviation and coefficient of variance is calculated. The mean current traces were then imported into PeakFit software for a baseline correction. The corrected traces were then exported and the original CV values were used to calculate standard deviations for the error. It should be noted that the aliquot of reporter used had experienced multiple freeze-thaw cycles. The data from the measurements with the original parameter set were noisy and were not fully analyzed. The location of this peak was different from previous experiments, where a peak was observed around 0.15 V. It is worth noting that the aliquot of reporter had experienced multiple freeze thaw cycles, so it may have degraded. As a control, LAMP buffer to determine the relative signal was also run. The LAMP buffer was already activated with magnesium in a previous experiment, then refrozen). The LAMP buffer was run neat, without any dilution. The signal from the LAMP buffer was much higher than the signal from the DETECTR reactions, as is visible in
The purpose of this experiment was to evaluate biotinylated gRNA functionality with Cas13a both in solution and immobilized on a surface. In this experiment three replicate runs of a biotin-modified gRNA (mod023) and three replicate runs of a non-biotin modified gRNA (R0003) were carried out. Three replicate “no target control,” or NTC runs were carried out for both the mod023 reporter and R0003 control. The procedure was carried out as follows:
The objective of this experiment was to verify if a 30 min incubation time was sufficient to produce a strong signal assay signal. Two concentrations were run. The procedure used is as follows:
Fluorescent quencher-based reporters were tested in an immobilized DETECTR assay. Streptavidin functionalized plates and biotin labeled reporters were used.
Results are illustrated in
The objective of this experiment was to determine whether binding both the gRNA and reporter to a plate allows the DETECTR assay to be as effective as binding the CAS protein-gRNA complex and reporter. This removes the need to functionalize the surface with the pre-complex of gRNA and CAS protein, allowing for an easier manufacturing process. Additionally, greater specificity can be achieved by allowing for more stringent washes. The following procedure was used.
The results of this experiment are shown in
The purpose of this experiment was to demonstrate target discrimination for immobilized reporters for the DETECTR reaction. The experiment design used in this experiment is shown in
Results are shown
The purpose of this experiment was to test the functionality of a one-pot reaction composed of both RT-LAMP and Cas12 variant enzyme-based DETECTR master mixes using SEQ ID NO: 17. Both master mixes were co-lyophilized together into one master mix. The reactions are functionally incompatible due to optimal reaction temperature differences, so they were evaluated independently. The RT-LAMP master mix shows reaction characteristics like the liquid controls. Results are shown for the RT-LAMP assay in
The purpose of this experiment was to investigate Cas14a1 which is functional at higher temperatures than RT-LAMP assay requires. In this example the temperatures were run up to 55° C. The results shown in
The purpose of this experiment was to investigate the performance of pooled and lyophilized master mixes containing both RT-LAMP and DETECTR reaction reagents together. Further this study showed that the pre-lyophilized mixture is stable for two weeks prior to lyophilization. The master mix of RT-LAMP and Cas12 variant-based DETECTR lyophilized together, was functionally tested separately using SEQ ID NO: 17, since the Cas12 variant (SEQ ID NO: 17) is not compatible with the RT-LAMP amplification conditions. These data show robust RT-LAMP and Cas12 variant-based DETECTR activity using SEQ ID NO: 17, comparable to the master mix that was stored at 4° C. for two weeks prior to lyophilization. Results are shown in
Previous to this example, lyophilized sample volume was demonstrated at 250 μL and performed in glass vials. This example provides data for lyophilization of 25 μL samples in 8-well plastic strip tubes. Additionally, the pre-lyophilization (3 weeks at 4° C.) and values are comparable showing stability of the master mix as seen in
In this experiment, the performance of the one-pot DETECTR assay in a handheld microfluidic device was evaluated. Here, one-pot refers to both the RT-LAMP and DETECTR reagents lyophilized as one master mix of reagents. The functions performed on the handheld, microfluidic device included: sample intake; RNA extraction from sample; mixing of the sample with CRISPR reactants; and the transfer of the mixture to a well to heat the mixture to the reaction temperature. In the device, the mixture was heated, and fluorescence was monitored continuously over time.
The purpose of this example is to demonstrate a DETECTR™-based multiplexed assay using a lateral flow assay (LFA) strip for parallel readout as illustrated in
The purpose of this example is to demonstrate a lateral flow assay strip workflow utilizing a multiplex “Hotpot” assay as illustrated in
The purpose of this example is to demonstrate horse radish peroxidase (HRP) paper-based detection as illustrated in
The purpose of this example is to demonstrate an HRP-signal enhanced multiplexed lateral flow assay as illustrated in
The purpose of this example is to demonstrate multiplexed target nucleic acid detection utilizing Cas13 RNA cleaving specificity over DNA, HRP-signal enhancement, and capture oligo probe specificity as shown in
The purpose of this example is to demonstrate horse radish peroxidase (HRP) paper-based detection as illustrated in
The purpose of this example was to demonstrate an HRP and DETECTR™-based assay. In this example, reporters were cleaved by a Cas complex, or a DNAse enzyme in solution. The cleaved reporter was reacted to HRP-T20-biotin (SEQ ID NO: 1498). The supernatant solution was then added to a reaction volume that contained TMB and H2O2 to generate a color signal. The cleaved reporter-HRP conjugate was then detected by optical density measurement of the solution. Optical density measurements were acquired from the beginning of the reaction. The experiment was performed in two sets comprising 2 runs each, where each set was run 1 week apart. DNase and DETECTR™ were used separately in each run of each set. In the DNase runs, 1 nM of HRP target oligo was used in the filled in circle series. Results are shown in
Guide RNAs that were designed to bind to a different region within a single target nucleic acid were pooled as a strategy for enhancing the target detection signal from DETECTR assays. For examples, in this strategy, each DETECTR™ reaction contained a pool of CRISPR-Cas RNP complexes each of which targeted a different region within a single molecule. As discussed in the paragraphs below, this strategy resulted in increased sensitivity to target detection by using increased number of complexes/single target such that the signal is strong enough to detect within a Poisson distribution (sub-one copy/droplet) and provide a quantitative evaluation of target numbers within a sample.
To test the effect of guide pooling on target detection using the Cas12a nuclease, first, a Cas12a complexing mix was prepared. The R1965 (off-target guide), R1767, R3164, R3178 guides were present in the Cas12a complexing mix in either a pooled-gRNA format (a pool of two or more of the three guides selected from R1767, R3164, or R3178) or in a single-gRNA format (wherein R1767, R3164, R3178 were present individually) and the mix was incubated for 20 minutes at 37° C. A 2-fold dilution series for the template RNA (GF184) was created from a starting dilution concentration (wherein 5.4 μl of GF184 at 0.1 ng/uL was added to 44.6 μl of nuclease-free water). DETECTR master mixes which included the Cas12 complex, Reporter substrate, Fluorescein, Buffer, and diluted template (GF184 or off-target template GF577) were then assembled as shown in Table 7. The DETECTR mixes were then loaded into a Stilla Sapphire chip and placed into the Naica Geode. Crystals were created from thousands of droplets from each sample. No amplification step was performed. The signal from the Sapphire chips was measured in the Red channel. The results of the DETECTR assay showed enhanced Cas12a-based detection of the GF184 target using a pooled-guide format compared to DETECTR Cas12a-based assay using an individual guide format. For example, the DETECTR assays showed an enhanced signal from chamber 5 containing a pool of two guides R1767 and R3178, compared to the signal from chamber 2 or chamber 4 which contained the R1767 and R3178 in individual guide format, respectively, as shown in
Table 7 displays conditions, copies per chamber, number of droplets and copies/droplet per chamber.
Enhanced sensitivity to target detection with guide-pooling was observed in the case of Cas13a nuclease also. In these assays, a Cas13a complexing mix was prepared wherein the R002 (off-target guide), R4517, R4519, R4530 guides were present in either a pooled-gRNA format (a pool of two or more of the three guides R4517, R4519, and R4530) or single-gRNA format (wherein R4517, R4519, and R4530 were present individually) and the mix was incubated for 20 minutes at 37 C. DETECTR master mixes which included the Cas13a complex, FAM-U5 Reporter substrate, Buffer, and diluted template SC2 RNA (or off-target template 5S-87) was then assembled as shown in Table 8. The DETECTR mixes were then loaded into a Stilla Sapphire chip and placed into the Naica® Geode system. Crystals were generated from the droplets from each of the samples and incubated at 37° C. (no amplification step was performed). The signal from the Sapphire chips was measured in the Cy5 channel. The results of the DETECTR assay showed enhanced Cas13a-based detection of the SC2 target RNA using a pooled-guide format compared to a Cas13a-based detection of the SC2 target RNA using a single-guide format. For example, the DETECTR assays showed an enhanced signal from chamber 8 (saturated—not displayed), containing the template at a concentration of 1×106 copies, and a pool of the three guides R4517, R4519, and R4530, compared to the signal from chamber 2, chamber 4, or chamber 6 which contained the template at a concentration of 1×106 copies, and the guides R4517, R4519, and R4530 in individual guide format, respectively, as shown in
Table 8 displays conditions, copies per chamber, number of droplets per chamber and copies per droplet per chamber.
Next, the sensitivity of a target detection in Cas13a digital droplet DETECTR assays containing guide RNA in either a pooled-guide format versus a single guide format was assayed. DETECTR reaction master-mixes was prepared for each gRNA (R4637, R4638, R4667, R4676, R4684, R4689, R4691, or R4785 (RNaseP)) and included, in addition to the gRNA, the Cas13a nuclease, and the reporter substrate. After complexing, 2 μL of each RNP was combined in either a pooled-gRNA format (a pool of the seven gRNAs, i.e., R4637, R4638, R4676, R4689, R4691, R4667, and R4684) or remained in the single-gRNA format (wherein R4667, R4684, and R4785 (RNAse P were present individually). The template RNAs (Twist SC2, ATCC SC2, and 5s-87 off-target) were diluted to obtain a series of template concentrations. DETECTR reactions directed to the detection of the template RNAs (Twist SC2, ATCC SC2, and 5s-87 off-target template RNAs) were assembled by combining the Cas13a-gRNA RNPs with the diluted template RNA from the previous step as shown in Table 9. The assembled DETECTR reactions were loaded into chambers on a Stilla Sapphire Chip. The Chips were placed into the Naica® Geode system and crystals were generated using the droplet generation program and imaged to reveal droplets that contain detected targets.
The sensitivity of target detection by the DETECTR assays containing the pooled guides (R4637, R4638, R4667, R4676, R4684, R4689, R4691) was compared with the sensitivity of target detection by the DETECTR assays containing the single guides R4684, R4667, R4785 (RNAseP guide) in individual format. Relative quantification performed by counting the number of these positive droplets showed that the samples containing the pooled guide RNAs generated more crystals containing the amplified products per copy of starting target RNA than the samples containing the guide RNAs in individual format as shown in
Relative quantification of the number of droplets containing amplified target (per copy of starting target RNA) observed in chamber 5 (containing the seven-guide pool and the ATCC SC2 template RNA) is higher than the number of droplets observed in chamber 6 (containing the gRNA R4684 in individual format and the ATCC SC2 RNA), the number of droplets observed in chamber 8 (containing the control RNaseP gRNA in individual format with the ATCC SC2 template RNA) and the number of droplets observed in chamber 12 (containing the seven pooled gRNAs with no template RNA) as shown in
Table 9 displays the guide pool and template per chamber.
The following describes experiments carried out to determine limit of detection for HotPot reactions using Cas.M21526. The HotPot DETECTR-based assays were run using the protocol shown in
These experiments were conducted to identify potential additives for HotPot assays. 96 potential additives were identified and individually screened for their influence on the output fluorescence.
Additives that were identified to increase the speed of the reaction under the conditions tested included: betaine monohydrate, acetamide, GABA, L-proline, beta-alanine, 6-aminohexanoic acid, urea, methylurea, ethylurea, hypotaurine, NDSB-256, and ammonium acetate.
Additives that were identified to increase the signal strength from the reaction under the conditions tested included: trehalose, xylitol, D-sorbitol, sucrose, and trimethylamine N-oxide dihydrate.
Further HotPot experiments were carried out to determining the effects of using glycerol free (GF) Bsm DNA polymerase and glycerol (G) containing Bsm DNA polymerase with or without 250 mM TMAO added.
These experiments were carried out to synthesize hydrogels containing immobilized reporters co-polymerized with a mixture of oligomers as described in
Reporter was covalently incorporated into PEG hydrogels during polymerization. A 2:1 ratio mixture of unfunctionalized PEG (MW=600 monomers) and PEG-diacrylate (MW=700 monomers) were mixed together with a photoinitiator (2-Hydroxy-2-methylpropiophenone (Darocur 1173)) and 100 μM of Acrydite-modified Reporter 172 (/5Acryd/TTT TTT TTT TTT TTT TTT TT/16-FAMK//3Bio/(SEQ ID NO: 1482)). The mixture was exposed to UV light (365 nm, 200 ms) under a photomask. The mask was configured to polymerize the mix into circular cross-sectional rods of hydrogel 400 μm in diameter. Excess material was washed off hydrogels after polymerization. The acrydite group on the 5′ end of the reporter was covalently reacted with the acrylate groups of PEG-diacrylate oligomers during co-polymerization in order to incorporate the reporter into the hydrogel.
OnePot (using a Cas 12 enzyme) and HotPot (using Cas14a.1) DETECTR reactions were run as described herein by applying the programmable nuclease complexes and target nucleic acids to a tube containing the hydrogels. 6 hydrogels/reaction were added for OnePort DETECTR and 10 hydrogels/reaction for Cas14a. 1 HotPot DETECTR assays. DETECTR reactions were run for 60 min at 37° C. with mixing for Cas 12 OnePot or 60 min at 55° C. with mixing for Cas14a.1 HotPot. Duplicate reactions were run for each of a target RNA and the NTC for both Cas 12 OnePot and Cas14a. 1 HotPot.
The tubes were then spun down and the supernatant was applied to lateral flow strips. The sample pad of lateral flow strip contained anti-FITC conjugate particles (colloidal gold). If target was present, the supernatant contained cleaved FAM-biotin-labeled reporter molecules which bound to an anti-biotin (e.g., streptavidin) target line on the lateral flow strip. The anti-FITC conjugate particles bound the FAM moiety on the reporter molecules and a target band appeared on lateral flow strips at the anti-biotin target line. If target was not present (as in NTC DETECTR reactions), the supernatant did not contain any FAM-biotin-labeled molecules and nothing bound to the anti-biotin target line. The lateral flow assay strip also contained an anti-IgG flow control line, downstream of the anti-biotin target line, which bound to the anti-FITC moiety of the conjugate particles to confirm that the lateral flow assay functioned properly.
This example demonstrates a method of making and using a hydrogel comprising immobilized guide nucleic acids and reporters.
Guide nucleic acids are covalently incorporated into PEG hydrogels during polymerization. A 2:1 ratio mixture of unfunctionalized PEG (MW-600 monomers) and PEG-diacrylate (MW=700 monomers) are mixed together with a photoinitiator (2-Hydroxy-2-methylpropiophenone (Darocur 1173)), 100 μM of Acrydite-modified Reporter 172 (/5Acryd/TTT TTT TTT TTT TTT TTT TT/16-FAMK//3Bio/(SEQ ID NO: 1482)), and 100 μM of Acrydite-modified guide nucleic acids (e.g., R1763 with acrydite modification: /5 Acryd/UAA UUU CUA CUA AGU GUA GAU CCC CCA GCG CUU CAG CGU UC (SEQ ID NO: 1483), or R1965 with acrydite modification:/5Acryd/rUrArAr UrUrUr CrUrAr CrUrAr ArGrUr GrUrAr GrArUr UrUrAr CrArUr GrGrCr UrCrUr GrGrUr CrCrGr Ar G (SEQ ID NO: 1484)). The mixture is exposed to UV light (365 nm, 200 ms) under a photomask. The mask is configured to polymerize the mix into circular cross-sectional rods of hydrogel 400 μm in diameter. Excess material is washed off hydrogels after polymerization. The acrydite group on the 5′ end of the reporters and the acrydite group on the 5′ end of the guide nucleic acids are covalently reacted with the acrylate groups of PEG-diacrylate oligomers during co-polymerization in order to incorporate the reporters and the guide nucleic acids into the hydrogel.
OnePot (using a Cas12 enzyme) and HotPot (using Cas14a.1) DETECTR reactions are run as described herein by applying the programmable nucleases and target nucleic acids to a tube containing the hydrogels. 6 hydrogels/reaction are added for OnePort DETECTR and 10 hydrogels/reaction for Cas14a. 1 HotPot DETECTR assays. DETECTR reactions are run for 60 min at 37° C. with mixing for Cas 12 OnePot or 60 min at 55° C. with mixing for Cas14a. 1 HotPot. Duplicate reactions are run for each of a target RNA and the NTC for both Cas 12 OnePot and Cas14a.1 HotPot.
The tubes are then spun down and the supernatant is applied to lateral flow strips. The sample pad of lateral flow strip contains anti-FITC conjugate particles (colloidal gold). If target is present, the supernatant contains cleaved FAM-biotin-labeled reporter molecules which bind to an anti-biotin (e.g., streptavidin) target line on the lateral flow strip. The anti-FITC conjugate particles bind the FAM moiety on the reporter molecules and a target band appeared on lateral flow strips at the anti-biotin target line. If target is not present, the supernatant does not contain any FAM-biotin-labeled molecules and nothing binds to the anti-biotin target line. The lateral flow assay strip also contains an anti-IgG flow control line, downstream of the anti-biotin target line, which binds to the anti-FITC moiety of the conjugate particles to confirm that the lateral flow assay functions properly.
This example demonstrates a method of making and using a hydrogel comprising immobilized programmable nucleases, guide nucleic acids, and reporter.
Guide nucleic acids are covalently incorporated into PEG hydrogels during polymerization. A 2:1 ratio mixture of unfunctionalized PEG (MW=600 monomers) and PEG-diacrylate (MW=700 monomers) are mixed together with a photoinitiator (2-Hydroxy-2-methylpropiophenone (Darocur 1173)), 100 μM of programmable nuclease (e.g., Cas12 or Cas14a.1), 100 μM of Acrydite-modified Reporter 172 (/5Acryd/TTT TTT TTT TTT TTT TTT TT/16-FAMK//3Bio/(SEQ ID NO: 1482)), and 100 μM of Acrydite-modified guide nucleic acids (e.g., R1763 with acrydite modification:/5Acryd/UAA UUU CUA CUA AGU GUA GAU CCC CCA GCG CUU CAG CGU UC (SEQ ID NO: 1483), or R1965 with acrydite modification:/5Acryd/rUrArAr UrUrUr CrUrAr CrUrAr ArGrUr GrUrAr GrArUr UrUrAr CrArUr GrGrCr UrCrUr GrGrUr CrCrGr Ar G (SEQ ID NO: 1484)). The mixture is exposed to UV light (365 nm, 200 ms) under a photomask. The mask is configured to polymerize the mix into circular cross-sectional rods of hydrogel 400 μm in diameter. Excess material is washed off hydrogels after polymerization. The acrydite group on the 5′ end of the reporters and the acrydite group on the 5′ end of the guide nucleic acids are covalently reacted with the acrylate groups of PEG-diacrylate oligomers during co-polymerization in order to incorporate the reporters and the guide nucleic acids into the hydrogel. The programmable nucleases are immobilized by complexing with guide nucleic acids.
OnePot and HotPot DETECTR reactions are run as described herein by applying target nucleic acids to a tube containing the hydrogels. 6 hydrogels/reaction are added for OnePort DETECTR and 10 hydrogels/reaction for HotPot DETECTR assays. DETECTR reactions are run for 60 min at 37° C. with mixing for OnePot or 60 min at 55° C. with mixing. Duplicate reactions are run for each of a target RNA and the NTC for both OnePot and HotPot.
The tubes are then spun down and the supernatant is applied to lateral flow strips. The sample pad of lateral flow strip contains anti-FITC conjugate particles (colloidal gold). If target is present, the supernatant contains cleaved FAM-biotin-labeled reporter molecules which bind to an anti-biotin (e.g., streptavidin) target line on the lateral flow strip. The anti-FITC conjugate particles bind the FAM moiety on the reporter molecules and a target band appeared on lateral flow strips at the anti-biotin target line. If target is not present, the supernatant does not contain any FAM-biotin-labeled molecules and nothing binds to the anti-biotin target line. The lateral flow assay strip also contains an anti-IgG flow control line, downstream of the anti-biotin target line, which binds to the anti-FITC moiety of the conjugate particles to confirm that the lateral flow assay functions properly.
This example demonstrates a positive feedback loop system for amplifying the signal for each target nucleic acid molecule in a sample as described in
A mixture comprising programmable nucleases (15101), primary target-specific guide nucleic acids (15102), and primary target nucleic acids (15103) is flowed over a well. The well comprises two types of hydrogels. The first type of hydrogel (15105) contains secondary target-specific guide nucleic acids (15104) that are each immobilized by a single-stranded DNA molecule (15106) onto or into the first hydrogel (15105). The second type of hydrogel (15109) contains double stranded secondary target nucleic acids (15108) immobilized onto or into the second hydrogel (15109). The first programmable nuclease is complexed to the primary target-specific guide nucleic acid (15102). The second programmable nuclease is not bound to a guide nucleic acid.
The first programmable nucleases complexed with the primary target guide nucleic acids (15102) binds to the primary target nucleic acids (15103) to create activated programmable nuclease complexes that trans-cleave nearby single-stranded nucleic acid species. The species include reporters (15111), e.g., free-floating in solution or immobilized to a substrate or hydrogel e.g., adjacent the single-stranded DNA molecules (15106) and/or secondary target nucleic acids, as described herein and the single-stranded DNA molecules (15106) immobilizing the secondary target-specific guide nucleic acids (15104) on or in the first hydrogel (15105). Cleavage of the single-stranded DNA linkers (15104) releases the secondary target-specific guide nucleic acids (15104) from the first hydrogel (15105). This leads to a free-floating population of secondary target-specific guide nucleic acids (15104) in solution. The free-floating secondary target-specific guide nucleic acids (15104) complex with the second programmable nucleases. The second programmable nucleases are then able to bind to the secondary target nucleic acids (15108) immobilized on the second hydrogel (15109) to form activated programmable nuclease complexes that trans-cleave nearby single-stranded nucleic acid species, including reporters (15111) and the single-stranded DNA molecules (15106) on the first hydrogel (15105). This in turn leads to more additional cleavage of reporters (15111) and secondary target-specific guide nucleic acids (15104) in solution, which in turn goes on to create more activated programmable nuclease complexes that release further secondary target-specific guide nucleic acids (15104) into the solution, and so on, thereby amplifying the initial signal from the primary target nucleic acids.
The immobilized nucleic acids (guide nucleic acids, secondary target nucleic acids) comprise fluorescent moieties and quencher moieties. Cleavage of the immobilized nucleic acids separates the quencher moieties from the fluorescent moieties, thereby allowing the fluorescent moieties to produce a detectable fluorescent signal. The fluorescent moieties remain bound to the hydrogels (15105, 15109) after cleavage, which results in an increase in fluorescence at the hydrogel locations. Thus, the amplified signals can be detected by an optical instrument.
This example demonstrates a handheld device comprising an integrated sodium acetate LAMP reaction consumable handheld device. The purpose of the handheld device is to provide a heating source for programmable nuclease-based detection applications that do not allow for battery or other electrical power supplies. The integrated sodium acetate LAMP reaction consumable provides heating to LAMP and programmable nuclease-based reactions via heat released from the crystallization of liquid-phase sodium acetate. The design of the integrated sodium acetate LAMP reaction consumable allows for uniform heating of the LAMP and programmable nuclease-based reactions by uniformly surrounding the LAMP reaction with sodium acetate heating agent in a heating reservoir.
In the integrated sodium acetate LAMP reaction consumable handheld device, liquid-phase sodium acetate is stored in a heating reservoir as shown in
This example demonstrates compositions and methods for HotPot reactions using thermostable inorganic pyrophosphatase (TIPP) to improve signal generation. TIPP is an enzyme that can catalyze the hydrolysis of inorganic pyrophosphates. This example demonstrates compositions and methods for HotPot reactions using thermostable inorganic pyrophosphatase (TIPP) to amplify output signals.
HotPot reactions were carried out under with various compositions as shown in Table 10. In the Experimental Condition, the HotPot reaction was carried out in a solution comprising both TIPP and target nucleic acid. In Control A, the HotPot reaction was carried out with target nucleic acid but without TIPP. In Control B, HotPot reaction was carried out without target RNA. In Control C, HotPot reaction was carried out without TIPP and without target RNA.
Briefly, in a first experimental example, Cas14a effector proteins were complexed with sgRNA for 30 minutes at 37° C. The 1× concentration of proteins was 40 nM and the final concentration of sgRNA was 40 nM. 1 uL of the Cas14a complexing reaction was combined with the HotPot components listed in Table 9 for each experimental or control condition listed. Reactions were carried out at 55° C. for 60 minutes. Trans cleavage activity was detected by fluorescence signal upon cleavage of a fluorophore-quencher reporter in the HotPot DETECTR reaction.
In a second experimental example, a 2:1 ratio mixture of unfunctionalized PEG (MW=600 monomers) and PEG-diacrylate (MW=700 monomers) were mixed together with a photoinitiator (2-Hydroxy-2-methylpropiophenone (Darocur 1173)) and 100 μM of Acrydite-modified “Rep172” reporter (/5Acryd/TTT TTT TTT TTT TTT TTT TT/16-FAMK//3Bio/(SEQ ID NO: 1482)). The mixture was exposed to UV light (365 nm, 200 ms) under a photomask to generate circular cross-section rods of hydrogel containing immobilized reporters. Cas14a effector proteins were complexed with sgRNA for 30 minutes at 37° C. The 1× concentration of proteins was 40 nM and the final concentration of sgRNA was 40 nM. 5 μL of these RNPs was combined with the following components for a final volume of 50 μL (listed at final concentration): 10 μL of target RNaseP RNA (45 pg/uL) or no target control (“(−)”), reporter-immobilized hydrogels (10 hydrogels/uL), IB15 one pot LAMP trans-cleavage buffer, dNTPs (1 mM), RNAse inhibitor, Bsm DNA polymerase, Warmstart RTx reverse transcriptase, RNase P LAMP primer mix, and TIPP (0.5 U/uL) or water (“no TIPP”). Reactions were carried out at 55° C. for 35 minutes. Trans cleavage activity was detected with lateral flow assay strips. The supernatant for each reaction was applied to the sample pad of a lateral flow assay strip containing anti-FITC conjugate particles (colloidal gold). If trans cleavage occurred, the supernatant contained cleaved FAM-biotin-labeled reporter molecules which bound to an anti-biotin (e.g., streptavidin) target line on the lateral flow strip. The anti-FITC conjugate particles bound the FAM moiety on the reporter molecules and a target band appeared on lateral flow strips at the anti-biotin target line. If trans cleavage did not occur (as in NTC or no guide RNA reactions), the supernatant did not contain any FAM-biotin-labeled molecules, and nothing bound to the anti-biotin target line. The lateral flow assay strip also contained an anti-IgG flow control line, downstream of the anti-biotin target line, which bound to the anti-FITC moiety of the conjugate particles to confirm that the lateral flow assay functioned properly. Lateral flow strips without TIPP are shown in
Without being bound to any particular theory, it is believed that TIPP may be responsible for processing phosphates which are produced when the LAMP reaction uses up dNTPs. Those phosphates can take Mg2 ions out of solution, which may be cause a Cas enzyme to exhaust the remaining Mg2 quickly and cause the reaction to plateau more rapidly than would occur with additional Mg2 in the system. With the presence of TIPP, those phosphates may be processed and the Mg2+ may be released for use by a Cas enzyme, which can improve DETECTR signal strength as the Cas enzyme is able to react unhindered by a lack of Mg2.
The results show that the addition of TIPP improved signal compared to without TIPP and made detection on lateral flow strips more robust. The amount of TIPP added can be varied to allow flexibility in assay designs.
This example describes isothermal nicking enzyme pre-amplification for DETECTR reactions. CRISPR-based diagnostic DETECTR reactions using loop-mediated isothermal amplification (LAMP) can be completed in as little as 30 minutes. In some cases, longer LAMP amplification may be required to achieve single copy sensitivity. A DETECTR reaction utilizing nicking enzyme amplification (NEAR) was developed to reduce amplification time relative to methods utilizing LAMP amplification. The DETECTR reaction with NEAR also showed improved sensitivity over NEAR reactions performed with a beacon. In the DETECTR reaction with NEAR, a 30 to 40 nucleotide region of a target nucleic acid was amplified using NEAR and detected using a Cas12 variant (SEQ ID NO: 17).
NEAR amplification was performed by contacting the target nucleic acid with a forward primer, a reverse primer, dNTPs, a DNA polymerase, and a nicking endonuclease. The reaction was performed at 60° C. for 10 minutes then cooled to 4° C. The forward primer contained a 16 to 20 nucleotide region reverse complementary to a first strand of the target nucleic acid and a 16 to 20 nucleotide nicking enzyme stabilization, binding, and recognition site 5′ of the region reverse complementary to the target nucleic acid. The reverse primer contained a 16 to 20 nucleotide region reverse complementary to a second strand of the target nucleic acid and a 16 to 20 nucleotide nicking enzyme stabilization, binding, and recognition site 5′ of the region reverse complementary to the target nucleic acid. A NEAR duplex incorporating a nicking enzyme stabilization, binding, and recognition site into an amplicon also containing the region of the target nucleic acid was then generated, as illustrated in
This example describes detection of Streptococcus pyogenes using NECTR (NEAR combined with DETECTR) via amplification of the Strep-A target. The Strep-A target was amplified using the process described in Example 31. Specific conditions were optimized for Strep-A detection.
A 10 μL preamplification reaction was prepared at a final concentration of 1× Isothermal Amplification® Buffer (NEB), 1×NEBuffer™ 3.1 (NEB), 300 nM dNTPs (NEB), 4U of Bst 2.0, 3U of Nt BstNBI, 500 nM of the forward primer (SEQ ID NO: 79), and 100 nM of the reverse primer (SEQ ID NO: 80). In order to prevent non-specific amplification, the primers were added last.
8 μL of the prepared pre-amplification mixture was dispensed into a 96-well or 384-well plate. 2 μL of the target nucleic acid was added to each well in order to prevent early initiation of the reaction. Sample mixtures are mixed thoroughly and centrifuged to ensure that mixture is at the bottom of the wells. The pre-amplification reactions were incubated at 60° C. for 10 minutes and immediately placed at 4° C. or on ice to stop the reaction.
Detection of the Strep-A was achieved by our DETECTR technology via Cas12a. A Cas12a complexing reaction was prepared at a final concentration of 1× MB3, 160 nM of Cas12 variant (SEQ ID NO: 17) and 160 nM of R1107 crRNA (SEQ ID NO: 76) and incubated at 37° C. for 30 minutes. After incubation, the Beacon-AlexaFluor 594 (SEQ ID NO: 86) was added to the complexing reaction at a final concentration of 400 nM.
In a 384-well black assay plate on ice, 13 μL of the 1× MB3 and 5 μL of the Cas12a complexing mixture was added. Using aseptic technique to prevent any nucleic acid contamination, 2 μL of the pre-amplification reaction was added to the assay plate. The plate was sealed with an optically clear adhesive and spun at 2000 rcf. The plate was read at AF594 setting with extended gain.
This example describes detection of SARS-CoV-2 using NECTR (NEAR combined with DETECTR) via amplification of the E-gene target. The Strep-A target was amplified using the process described in Example 31. Specific conditions were optimized for E-gene detection.
A 10 μL preamplification reaction was prepared at a final concentration of 1× Isothermal Amplification Buffer (NEB), 1×NEBuffer 3.1 (NEB), 300 nM dNTPs (NEB), 4U of Bst 2.0, 3U of Nt BstNBI, 500 nM of the Forward primer (SEQ ID NO: 35), and 100 nM of the reverse primer (SEQ ID NO: 36). In order to prevent non-specific amplification, the primers were added last.
8 μL of the prepared pre-amplification mixture was dispensed into a 96-well or 384-well plate. 2 μL of the target nucleic acid was added to each well in order to prevent early initiation of the reaction. Sample mixtures are mixed thoroughly and centrifuged to ensure that mixture is at the bottom of the wells. The pre-amplification reactions were incubated at 60° C. for 10 minutes and immediately placed at 4° C. or on ice to stop the reaction.
Detection of the Strep-A was achieved by our DETECTR technology via Cas12. A Cas12 complexing reaction was prepared at a final concentration of 1× MB3, 160 nM of Cas12 Variant (SEQ ID NO: 17), and 160 nM of R1107 crRNA (SEQ ID NO: 76) and incubated at 37° C. for 30 minutes. After incubation, the Beacon-AlexaFluor 594 (SEQ ID NO: 81) was added to the complexing reaction at a final concentration of 400 nM.
In a 384-well black assay plate on ice, 13 μL of the 1× MBuffer 3 and 5 μL of the Cas12a complexing mixture was added. Using aseptic technique to prevent any nucleic acid contamination, 2 μL of the pre-amplification reaction was added to the assay plate. The plate was sealed with an optically clear adhesive and spun at 2000×g rcf. The plate was read at AF594 setting with extended gain.
This example describes optimization of Mg2+ concentration for the NEAR reaction. In addition, the performance of Bst 2.0 and Bst 3.0 were assessed. In this example, the protocol used was identical to that used in Example 31.
After amplification of Strep-A, the samples were tested in different buffers, Thrmopol, IsoAmp® I, and IsoAmp® II (NEB) at different added Mg2+ concentrations-0 mM, 2.5 mM, 5 mM, 7.5 mM, and 10 mM. The elapsed time at which it took to achieve a fluorescent readout from the Beacon plate reader was measured, with a lower value indicating that there were more copies of the amplicon present in the solution.
It was found that no additional magnesium was required and that Bst 2.0 was preferred for the NEAR reactions under the conditions tested. As shown in
This example describes how to select a guide RNA for detection of Strep-A, but it will be understood that the teachings described here may be used to select guides for other targets of interest as desired. The same primers and probe used in Example 32 were used. The following experiment determined if ssDNA could be detected using DETECTR from a NEAR reaction. Due to the biased reaction, gRNAs were generated in the reverse direction to detect any ssDNA produced from the nicking occurring on the forward primer nicking site.
A panel of 19 gRNAs was synthesized in order to determine which gRNA worked best in the DETECTR reaction under the conditions tested. As shown in
This example describes quantification of NEAR RNA targets derived from SARS CoV-2 using the NECTR system described in Example 33. We used two NEAR primers to amplify the SARS-CoV-2 target sequence and a guide design as shown in
Next, we tried to determine the minimal incubation time of a NEAR reaction of Strep-A targets prior to detection via DETECTR. The NEAR protocol was substantially similar to that described in Example 32. Incubation of the target with NEAR reaction components proceeded for 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, or 6 minutes prior to DETECTR. Under the conditions tested, a minimum of 2 minutes was observed to be required to achieve a detectable DETECTR signal, however, 6 minutes was found to be more optimal as it achieved a signal much higher than the limit of detection, as shown in
This example describes a comparison of NEAR amplicon detection using orthogonal Cas system to detect the target. The protocol of Example 32 was used for this example. Here, we utilized a Cas12 variant (SEQ ID NO: 17), a Cas13 variant (SEQ ID NO: 21), and a Cas14 variant (SEQ ID NO: 3). They were chosen because they have different cleavage preferences for the reporter molecule and can be multiplexed together. In
We also optimized the stability of the hairpin loop in the nickase stabilization region present in the NEAR primers. Without being bound to any particular theory, it is thought that the hairpin loop may impact the nickase activity of the enzyme. To test this, we used, alternative primer pairs to destroy, maintain, or enhance the hairpin loop formation (
This example describes comparison of different reverse transcriptases for the RT-NECTR reaction described in Example 33. In this study, we tested Wartmstart RTx (NEB), Bst 3.0, and Omniscript RT (Qiagen) and they were compared by using the limit of detection as a metric.
Primer concentrations were optimized in order to determine which concentrations would result in a better signal using the RT-NEAR reaction described in Example 33. In
Due to the need to reverse transcribe the SARS-CoV2 prior to detection, different amplification times need to be tested in order to determine the optimal resulting signal from RT-NECTR protocol of Example 33. In
This example describes testing of different Cas systems for orthogonality of the NEAR-DETECTR reaction described in Example 32. Cas14 variant (SEQ ID NO: 3) has been previously reported to detect ssDNA, whereas Cas13 variant (SEQ ID NO: 21) has been determined to be capable of detecting ssDNA. Thus, it was determined whether it was possible for the Cas14 variant or Cas 13 variant to be used in NEAR-DETECTR to detect ssDNA. As shown in
The objective of the experiment described was to produce a visible signal with an HRP-based DETECTR assay, particularly in the absence of target nucleic acid amplification. In this experiment, reporters comprising HRP and biotin were immobilized onto beads functionalized with Streptavidin via the biotin-Streptavidin interaction. A schematic can be seen in
Reporter immobilization to the beads. The HRP-reporters with biotin functional groups were immobilized onto 10 μL of streptavidin beads at reporter concentrations of 1 nM, 250 nM and 500 nM respectively.
HRP-based DETECTR Assay. To each of the three separate bead suspensions, at 1 nM reporter, 250 nM reporter and 500 nM reporter, respectively, the programmable nuclease Cas13 having SEQ ID NO: 21, complexed with the nucleic acid guide, and 4 nM of target was added and allowed to react at 37° C. for 30 minutes. In this step, the HRP portion of the reporter was released into solution via cleaving by the activated programmable nuclease-guide nucleic acid-target complex. The supernatant containing the cleaved HRP was then decanted from the bead suspension and combined with a solution containing the HRP enzyme substrate, TMB, at an approximate ratio of 2:1 (v/v) TMB to HRP supernatant. The HRP supernatant was first transferred to a fresh 96-well clear plate and then TMB was added to each well at approximately T=0. It takes about 20-30s from adding the TMB to starting the absorbance read on the plate reader. While the HRP enzyme consumed the substrate, TMB, the absorbance of the solution was measured. Results can be seen in
An HRP-based DETECTR Assay was performed at six target concentrations to determine a limit of detection (LOD). Six aliquots were taken from the bead suspension functionalized with HRP reporter at 250 nM. A Cas13 programmable nuclease complexed with guide nucleic acid was added to all six. Target was added to each of five aliquots to obtain target concentrations of 700 attM, 7 fM, 70 fM, 700 fM and 350 μM. No target was added to the sixth aliquot to act as the no-target control (NTC). Reactions in all six aliquots were carried out at 37° C. for 30 minutes. The supernatant containing the cleaved HRP was then decanted from the bead suspensions and separately combined with solutions containing the HRP enzyme substrate, TMB, at an approximate ratio of 2:1 (v/v) TMB to HRP supernatant. While the HRP enzyme consumed the substrate, TMB, the absorbance of the solutions was measured. Results can be seen in
This example describes effects of different concentrations of reporter functionalized to a streptavidin-coated plate, and measurements to determine the limit of detection.
Reporter immobilization. The HRP-reporter, rep 194, as described herein, containing a biotin functional group was immobilized onto streptavidin plates in three separate aliquots, where the concentrations of reporter was 5 nM, 50 nM and 250 nM respectively. A schematic of the reporter can be seen in
HRP-based DETECTR Assay. To each of the three separate plates, at 5 nM reporter, 50 nM reporter and 250 nM reporter, respectively, a programmable nuclease Cas13, complexed with the nucleic acid guide, and 10 μM of target was added and allowed to react at 37° C. for 30 minutes. In this step, the HRP portion of the reporter was released into solution via cleaving by the activated programmable nuclease-guide nucleic acid-target complex. The supernatant containing the cleaved HRP was then decanted from the streptavidin-coated plates and combined with a solution containing the HRP enzyme substrate, TMB. While the HRP enzyme consumed the substrate, TMB, the absorbance of the solution was measured. Results can be seen in
An HRP-based DETECTR Assay was performed at five target concentrations to determine a limit of detection (LOD). Five wells of a streptavidin-coated plate were functionalized with HRP reporter at 250 nM. Cas13 programmable nuclease complexed with guide nucleic acid was added to all five. To each of four wells, target was added to obtain target concentrations of 10 fM, 100 fM, 1 μM and 10 pfM. No target was added to the fifth well to act as the no-target control (NTC). All five conditions were allowed to react with the HRP-reporter-bound streptavidin-coated plates at 37° C. for 30 minutes. The supernatant containing the cleaved HRP was then decanted from the plates and combined with solutions containing the HRP enzyme substrate, TMB. While the HRP enzyme consumed the substrate, TMB, the absorbance of the solutions was measured. Results can be seen in
This example describes an HRP-DETECTR assay utilizing HRP-hydrogels comprising different PEG-DA ratios.
HRP-Hydrogel Prep. Preparation of HRP-hydrogels was similar to that described in Example 25 above. 22.5 μL of PEG/PEG-DA monomer mix was added to a PCR tube with 2.5 μL of reporter for a final concentration of either 10 μM or 1 μM reporter in 25 μL hydrogel mix. The mixture was vortexed and spun down briefly, then pipetted into a PDMS microfluidic channel on a glass slide. The hydrogel mixture was polymerized by exposure to 365 nm UV light (275 ms for 20% PEG-DA, 260 ms for 35% PEG-DA). Hydrogels were collected from the PDMS channel, collected, washed, resuspended, spun down, and supernatant removed, leaving about 100 μL of hydrogel. Hydrogels were stored in 1 mL of wash buffer at 4° C. overnight. The concentration of each hydrogel pool was determined by pipetting 1 μL onto a glass slide and counting particles under a microscope. Volume was adjusted to reach the desired final concentration.
HRP-Hydrogel DETECTR assay. Detection assays were performed using different PEG-DA concentrations (20% or 35%) co-polymerized with different concentrations of reporters (10 uM or 1 uM) comprising HRP-based detection moieties and acrydite functionalities (rep188). Samples included 4 nM of target, a no-template control (NTC), and an off-target control (OTC). The OTC sample included a nucleic acid with a different sequence from the target nucleic acid, and served as a control for the specificity of the reaction in that no signal was expected in response to the OTC. One of the reactions did not include any reporter (a “no rep control”). Two replicates were run for each condition (“prep 1” and “prep 2”).
The reactions were run as follows. 5 μL of hydrogel were aliquoted into single PCR tubes. Programmable nuclease detection complexes were prepared by combining 3.6 μL of nuclease-free water, 1 μL of 5× buffer, 0.08 μL of gRNA, 0.32 μL of 5 μM Cas enzyme SEQ ID NO: 34, and incubating at 37° C. for 20-30 minutes. 26 μL buffer was added to the PCR tubes containing hydrogels, along with 5 μL of detection complexes, and 4 μL of sample (i.e., target, NTC, or OTC). The final concentrations of components in the reaction were 40 nM gRNA, 40 nM Cas enzyme, and 4 nM target. The mixtures were vortexed, spun down briefly, and incubated for 30 minutes at 50° C. with no shaking. The reactions were stopped by placing on ice. Tubes were spun down for 5 minutes, and 10 μL of supernatant from each tube was pipetted to separate wells of a 96-well plate. 20 μL of TMB was added to the wells, and absorbance was read on a plate reader. Results for absorbance are illustrated in
In this example, an HRP-DECTER assay is demonstrated by immobilizing HRP-reporter onto NHS-activated resin.
HRP-reporter immobilization to NHS-activated resin. Columns were prepared and the assay were run according to the schematic illustrated in
HRP-DETECTR assay. The detection reagents were prepared by combining 313.2 μL nuclease free water (NFW), 78.4 μL 5× buffer, 0.08 μL crRNA, 0.32 μL of 5 μM Cas enzyme SEQ ID NO: 34, and the resulting 392 μL were added to the resin in the columns. Columns then received either 8 μL of 50 nM target or NFW, and were incubated on a thermomixer at 48° C. for 45 minutes at 500 RPM with intermittent shaking (15 seconds on, 2 minutes off). The columns were spun down and eluate collected. The columns were washed four times with 400 μL buffer, and the wash fractions were collected. 150 μL of eluate was added to wells of a clear 96-well plate. 50 μL of substrate (TMB) was added to each well and absorbance (optical density at 650 nm) was measured. Illustrative results are shown in
This example describes an HRP-DETECTR assay the demonstrated 100 fM limit of detection utilizing a poly-HRP reporter.
Preparation of reporter resin. Carboxylic beads having a concentration of about 10 mg/mL were functionalized with a poly-HRP reporter, as illustrated in
HRP-DETECTR assay. Two different volumes of the reporter poly-HRP reporter conjugated beads were tested, 2 μL and 5 μL. A programmable nuclease having SEQ ID NO: 34 was complexed with guide nucleic acid and added to the beads. Reactions also included 10 fM, 100 fM, 0.5 pM, 1 pM, 10 pM, 100 pM or 1 nM of target, except for a no template control (NTC). The contents of the columns were then reacted for 45 minutes at 50° C. The liquid portion was then collected and added to a TMB solution. The absorbance of the mixture was measured over time, and results are illustrated in
This example describes a method of lysing a sample with a protease to release nucleic acids so that they are accessible to a programmable nuclease (e.g., in a DETECTR reaction).
A sample comprising cells and/or viral particles is exposed to a lysis buffer solution comprising Proteinase K to digest the proteins (including RNAses, DNAses, matrix proteins, viral proteins, etc.) in the sample and release any nucleic acids therefrom. The sample is incubated in the lysis buffer solution at 37° C. for a pre-determined digestion time (e.g., 1 hour) to allow for proteolysis to occur. A serine protease inhibitor (e.g., PMSF) is then added and the mixture is incubated at 37° C. for a pre-determined inhibition time (e.g., 1 hour) to inactivate the Proteinase K. The programmable nuclease and additional detection reagents (e.g., guide nucleic acids, reporters, etc.) are then added to a mixture comprising the released nucleic acids and inactivated Proteinase K.
This example describes a method of detecting a target nucleic acid using a programmable nuclease, a guide nucleic acid, a first reporter, an enzyme, and a second reporter. In general, formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid activates the programmable nuclease (a Cas protein, in this example). The activated Cas protein cleaves the first reporter, releasing a first nucleic acid section (a cyclic adenylate, in this example), which activates the enzyme (NucC, in this example). NucC then cleaves the second reporter, which separates a fluorescent label from a quencher. Detection of the fluorescent label is used as an indicator for presence of the target nucleic acid in the sample.
In this detection scheme, Cas13 removes a blocking group from a linear RNA (blocked-activator). The blocking group is removed specifically due to the cleavage preferences of Cas13 for U's over A's. The shorter activator is now able to function to activate NucC. NucC then cleaves a dsDNA reporter molecule which generates a fluorescent or colorimetric signal. The Cas13 is capable of multiple turnovers and so is the NucC. The activity of these two proteins together generates a signal amplification effect, where the initial Cas13 detection of a pathogen or other nucleic acid sequence of interest is finally reported out by NucC. An illustrative schematic of this system is illustrated in
The temperature tolerance of NucC was assessed in order to determine whether NucC retains its ability to cleave double-stranded DNA at temperatures above 37° C. and is compatible with DETECTR assays run at elevated temperatures with a thermostable programmable nuclease (e.g., a Cas13c, etc.). Cleavage assays were performed with Vibrio metoecus NucC (VmeNucC) in MB3 buffer with FQ double-stranded DNA reporter (rep213+214). The final concentration of activator (cyclic triadenylate activator) was 1 nM and the final VmeNucC concentration was 250 nM.
Each reaction was run for 60 minutes at a different temperature: 40° C., 45° C., 50° C., 55° C., 60° C., or 65° C. In parallel, a second series of reactions at the different temperatures were performed in the absence of the 1 nM activator. Graphical representations of the results with and without activator present during the reactions are shown in
Activation of NucC Orthologs with Uncapped Linear Activators
The activity of NucC after addition of a linear activator was assessed in order to determine whether NucC could be activated by linear oligos, in addition to cyclic oligos, as shown in
Each reaction was run for 240 minutes with or without the presence of the linear activator at various concentrations. Graphical representations of the results with and without linear activator present during the reactions are shown in
This example describes results for several approaches to signal amplification of Cas-based detection of target nucleic acids using horseradish peroxidase (HRP). Systems included those based on surface-tethered HRP, magnetic bead-tethered HRP, magnetic bead capture of HRP, and processes that included an HRP concentration step.
Surface Tethered Approach: HRP was bound to the surface of functionalized plates (e.g., streptavidin, NHS, epoxy, etc.) by a DNA or RNA tether; details are specified in the results section below. Wells were washed 4× with wash buffer. After the final wash, Cas reaction buffer was added to each well, along with the Cas-gRNA complex. Target was then added to the reaction wells. Reactions were allowed to proceed at 37° C. for 30 minutes. The supernatant was then transferred to a second plate which contained the HRP substrate, TMB. Absorbance was then measured for 30 minutes at 37° C.
Magnetic Bead Tethered Approach: This process was similar to the surface-tethered approach, but the HRP was bound to the surface of functionalized magnetic beads. After incubating with activated Cas-gRNA complex at 37° C. for 30 minutes, the magnetic beads were pelleted with a magnet, and the supernatant was transferred to a plate containing the HRP substrate, TMB. Absorbance was then measured for 30 minutes at 37° C.
Magnetic Bead Capture Approach: HRP-biotin reporter was incubated in-solution with activated Cas complex at 37° C. for 30 minutes. Magnetic beads were then added to capture the intact reporter and allow HRP reporters that had been cleaved by the Cas complex to remain in solution. The supernatant was then transferred to a plate containing the HRP substrate, TMB. Absorbance was then measured for 30 minutes at 37° C.
HRP Concentration Approach: Anti-HRP antibodies were attached to agarose resin and the resin was loaded into an empty micro-spin column. Following the Magnetic Bead Tethered Approach or Magnetic Bead Capture Approach, the HRP-containing supernatant was transferred to the prepared column and incubated at ambient temperature for 30 minutes. The column was centrifuged and the flow through was discarded. The HRP substrate, TMB, was added to the column and incubated for 30 minutes at room temperature and then recovered by centrifugation. Stop Solution was then added to the column and recovered by centrifugation. The recovered TMB and Stop Solution were combined and the absorbance was measured.
Surface-tethered reporters provide various advantages detailed herein. However, in some early experiments, detection below 1 pM (about 15 million copies per reaction) was challenging in some implementations. In an effort to improve sensitivity for lower target concentrations, we redesigned the assay so that the HRP reporter would be cleaved in-solution and then the reporter captured onto magnetic beads. Without wishing to be bound by theory, it was believed that the Cas protein would have easier access to reporter targets in solution than those bound to a surface. An example illustration of the assay workflow is provided in
We evaluated the impact of reporter concentration on the performance of the in-solution assay (
We also evaluated the choice of magnetic bead and the binding buffer for the reporter (
As an alternative to the in-solution HRP reporter followed by capture, we also evaluated the possibility of reducing potential steric inhibition of the reporter cleavage reaction by increasing the length of the tether to better accommodate the size of the Cas protein. Instead of using functionalized plates or slides, we used magnetic beads, as we found these easier to manufacture and also increased the surface area available for the Cas to interact with while active. Our experiment demonstrated that a shorter reporter than we used initially showed lower signal and lower sensitivity than a longer reporter that was designed to allow for better access of the Cas protein to the cleavable region (e.g., single strand RNA region) (
To evaluate the performance of the assay with the optimized reporter and bead concentrations, we performed a 22 replicate experiment with different concentrations of target nucleic acid, expressed in copies per reaction (“copies/rxn” or “cp/rxn”). Concentrations tested included 2,000,000 copies/rxn, 1,000,000 copies/rxn, 500,000 copies/rxn, and 0 copies/rxn, using ddPCR quantified synthetic SARS-CoV-2 RNA from Twist. Illustrative results are shown in
In the experiments discussed above in this example, the effective TMB and HRP concentrations were constrained by the volumes used for the DETECTR® reaction and the binding buffers. We hypothesized that capturing the HRP onto a surface after the cleavage reaction but before signal detection could provide better control over the TMB-HRP reaction kinetics.
This example demonstrates a positive feedback loop system for amplifying the signal for each target nucleic acid molecule in a sample. The assay used in this example is illustrated diagrammatically in
A mixture comprising a first programmable nuclease (21401, Cas #1) complexed with primary target-specific guide nucleic acids, a second programmable nuclease (21402, Cas #2) complexed with secondary target-specific guide nucleic acids immobilized to the surface, a secondary target nucleic acid (21403, target #2) immobilized to the surface, a reporter molecule in solution (21404), and finally, a primary target nucleic acid (21405, target #1) capable of interacting with the complex (21401, Cas #1) when introduced into solution. The programmable nuclease (21401, Cas #1) complex then trans-cleaves an ssDNA tether between the surface and the second programmable nuclease (21402, Cas #2) complex. This releases the second programmable nuclease (21402, Cas #2) into solution so it can recognize its corresponding target (21403, target #2) which activates the second programmable nuclease (21402, Cas #2) complex. The activation of the 21402 complex creates a feedback loop where further programmable nuclease 21402 can be released. Activated 21402 complex is also effective to transcollaterally cleave the reporter molecule. Cleavage of the immobilized nucleic acids separates the quencher moieties from the fluorescent moieties, thereby allowing the fluorescent moieties to produce a detectable fluorescent signal. In some embodiments, this assay can be conducted on hydrogels as described in Example 28. In some embodiments, this assay can be conducted using immobilized reporters as described in
Immobilized guide RNAs used in the assay to detect an immobilized secondary target (Target 2) were those shown in Table 13, with results shown in
This example describes production of a visible signal upon target nucleic acid detection, particularly in the absence of target nucleic acid amplification. In this experiment, reporters comprising HRP detection moieties linked to a tethered guide-reporter hybrid molecule (gREP) are immobilized to a surface by a linkage. The gREP forms a complex with a programmable nuclease, which is activated upon further complexing with a target nucleic acid. The activated programmable nuclease is effective to transcollaterally cleave one or more linkages connecting the HRP detection moiety to the remainder of the gREP. The HRP is released, and the supernatant containing the released HRP is then transferred to a secondary chamber containing an HRP substrate. When HRP is present in the supernatant it acts on the HRP substrate to produce a detectable signal. In some embodiments, the detectable signal can be detected by measuring an absorbance or fluorescence within the secondary chamber. A schematic representation of the assay is shown in
This example describes a single-step isothermal nicking enzyme amplification reaction (NEAR) for use with programmable nuclease-based detection reactions (e.g., DETECTR). The detection reaction incorporates CRISPR-based specificity with NEAR's rapid and robust amplification. A schematic of the NEAR amplification reaction can be found in
LAMP, another isothermal amplification method, may take around 20-45 minutes of incubation time for sufficient amplification of a target sequence at a low copy number, which may be too long of a process for some point-of-care diagnostics settings. This duration can be mitigated through the incorporation of nicking enzyme assisted amplification reactions such as EXPAR, NEAR, and NEMA. In at least some instances, NEAR may be ideal for isothermal single-step reactions (e.g., HotPot reactions) due to its robust amplification speed at one temperature, though optimization may be beneficial in view of closely positioned primer pairs, which may be prone to off target activation. This example describes results showing development of NEAR for use with an illustrative Cas-based detection assay (e.g., DETECTR), which is ideal for amplifying and detecting targets of interest rapidly in a single-step reaction.
Cas variants were tested for performance in a high temperature and salt environment compatible with a single-step NEAR-detection reaction. Two-step NECTR reactions using a Cas 12 variant having SEQ ID NO: 34, along with two other thermostable variants (a Cas12 variant having SEQ ID NO: 120, and a Cas12 variant having SEQ ID NO: 116) were performed and showed high activity when run in NEAR buffer conditions (
Next, one-step HotPot reactions were performed using the Cas 12 variant of SEQ ID NO: 34 and a Cas12 variant having SEQ ID NO:116 (
A NEAR reaction was thus performed using either the normal SARS-CoV-2 RNA template or the guide RNA as a template, with all other conditions the same as described previously with respect to
Given that the guide RNA itself could be used to trigger the NEAR reaction, new primer sets were designed around two alternative target sites, R8895 for influenza B virus (IBV) (
Three primer sets (25-24 and 25-26 for IBV, and 31-36 for RSV-A) were chosen for testing in the single-step based NECTR reaction due to their exhibiting high signal-to-noise (as demonstrated in
These reactions were next replicated with the addition of TIPP or in the absence of TIPP (
This example describes a method of lysing a sample with a protease to release nucleic acids so that they are accessible to a programmable nuclease (e.g., in a DETECTR reaction). This example describes the use of Savinase® (Subtilisin 309) in a universal prep buffer for removing nucleases and thus reducing endogenous RNase activity. Biological samples, including saliva, nasal fluid, sputum, and blood, typically contain endogenous RNase species. Programmable nuclease-based detection assays for RNA viruses on crude sample preps often fail due to the presence of these RNases, which can cause degradation of the naked RNA viral template when it is released from viral particles during sample preparation. As RNases are protein in nature, the use of proteases can remove them, however the high variety of different RNase species in a sample means that few proteases will be able to remove all RNases present in the sample. It may also be beneficial to inhibit native RNase activity, particularly when the sample will be used in a programmable nuclease-based detection reaction, in order to prevent degradation of added nucleases (e.g., CRISPR-Cas enzymes) by the native RNAses. However, such nucleases may themselves be targets of proteases, therefore any protease used for sample preparation should preferably be removed, denatured, or otherwise inactivated before the sample is exposed to programmable nuclease-based detection reagents and conditions (e.g., DETECTR reaction compositions).
In this example, Savinase®, a serine protease, was tested for activity against the RNases present in fresh saliva samples using an RNase Reporter Assay (described below), and resulted in significantly reduced RNase activity. Savinase® was shown to be inhibited by 10 mM PMSF (phenylmethylsulfonyl fluoride, protease inhibitor). 10 mM PMSF was found to have no significant inhibition of a DETECTR reaction run using CasM.26. De-salted Savinase® successfully reduced saliva RNase levels, so that after treatment, following the addition of PMSF and spike-in of SARS-CoV-2 synthetic RNA, DETECTR was able to produce a positive signal. Without Savinase® treatment, RNase activity in saliva resulted in DETECTR failure.
Savinase® was purchased from Sigma® (P3111). Savinase® was initially screened alongside other proteases for activity against RNases from fresh saliva (50% v/v) in 10 mM Tris pH8. Saliva was chosen as the test sample. An RNase reporter assay (0.01% v/v Rep132 in 10 mM Tris pH8, incubated at 37° C. for 1 hour on QS5 using Cy5 channel for detection every 25s) was used to test the RNase activity following incubation at 37° C., 55° C., or 65° C. with 5% protease (v/v) for 1 hour. Savinase® showed the lowest RNase activity, with the preferred incubation temperature being 65° C. This was then repeated using 1× MB1 (buffer), 5% v/v protease, incubation at 37° C., 55° C., or 65° C. for 1 hour, and RNase activity measured using the FQ RNA reporter Rep132 (5′-Alex647N/rUrUrUrUrU/IAbRQSp-3′) in MB1. Again, Savinase® showed the lowest RNase activity, with the preferred incubation temperature tested being 65° C.
Savinase® was then tested against the other proteases which demonstrated some RNase removal at comparable activity concentrations of 3U, where possible. The lot of Savinase® used was documented as having 18U/g and the density of Savinase® at 1.16 g/mL, indicating an activity of 0.02U/uL. 30 μL of Savinase® (corresponding to 0.63U) was used in a 100 μL reaction (50 μL saliva, 20 μL 5× MB1), at 37° C., 55° C., or 65° C. for 1 hour; followed by RNase Reporter Assay (described above). Despite the units of Savinase® used being ˜5-fold less than for many of the other enzymes tested, Savinase® still showed to clearly have the best RNase removal of the proteases tested. The protease inhibitor PMSF was then tested for activity against Savinase® following the incubation at 55° C. and 65° C. for 1 hour; at 0, 0.1 mM, 1 mM, and 10 mM in 50% isopropanol, at room temperature (no incubation time) and Pierce™ Protease Activity assay (Thermo Scientific® 23266), 25° C. for 45 m on QS5 with signal detected using FAM channel, measuring every 25s. 10 mM PMSF was shown to significantly reduce Savinase® activity.
Further testing of Savinase® alone and in combination with the next most RNase-removing protease (Protamex, Sigma® P0029) was carried out using different ratios of each enzyme combined. Savinase® was confirmed to have significant anti-RNase activity via the RNAse reporter assay described above (
Savinase® also showed significant reduction in Protease Activity Assay following inhibition with 10 mM PMSF (in 15% isopropanol) after just 1 minute at room temperature; this was reduced further with longer incubation times (5 minutes and 15 minutes) (
Protease inhibitors (including PMSF) were tested for activity against CasM.26 via a DETECTR reaction with 1.5M cp/rxn Twist SARS-CoV-2 RNA, R4684 guide RNA (5′-GCCACCCCAAAAAUGAAGGGGACUAAAACAAGAAGAAUUCAGAUUUUUAA-3′ (SEQ ID NO: 1517)), (62.5 nM final concentration), reporter Rep132 (100 nM final concentration), and buffer MB1. PMSF was shown not to adversely affect DETECTR (
Some commercially-purchased Savinase® was found to be non-compatible with DETECTR, unless in very dilute concentrations. It was hypothesized that salts in the commercially-purchased Savinase® may have a deleterious effect on the DETCTR reaction. To test this, commercially-purchased Savinase® was subjected to desalting to determine whether salt concentration may contribute to reduced activity of the DETECTR reaction. Desalting of Savinase® on PD10 columns was shown to remove calcium from the Savinase® via an Alginate Polymerization Assay.
Desalting resulted in a Savinase® solution which still retained protease activity, was still highly effective at removing RNase activity from saliva, and showed potential compatibility with RNA-based DETECTR. In an exemplary DETECTR reaction, saliva treated with PD10 desalted Savinase® is incubated at 65° C. for 5 minutes, denatured with 10 mM PMSF at room temperatures, spiked with 2M cp/rxn SARS-CoV-2 synthetic RNA (Twist), and then incubated with CasM.26, R4684 guideRNA, reporter Rep132, and MB1 buffer at 37° C. Fluorescence is monitored over time to determine whether reporter is cleaved by the Cas enzyme as described herein.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein can be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims priority to U.S. Provisional Application Ser. No. 63/213,642 filed on Jun. 22, 2021; U.S. Provisional Application Ser. No. 63/222,377 filed on Jul. 15, 2021; U.S. Provisional Application Ser. No. 63/225,284 filed on Jul. 23, 2021; U.S. Provisional Application Ser. No. 63/231,536 filed on Aug. 10, 2021; U.S. Provisional Application Ser. No. 63/239,866 filed on Sep. 1, 2021; U.S. Provisional Application Ser. No. 63/239,917 filed on Sep. 1, 2021; U.S. Provisional Application Ser. No. 63/241,952 filed on Sep. 8, 2021; U.S. Provisional Application Ser. No. 63/245,610 filed on Sep. 17, 2021; U.S. Provisional Application Ser. No. 63/246,754 filed on Sep. 21, 2021; U.S. Provisional Application Ser. No. 63/255,356 filed on Oct. 13, 2021; U.S. Provisional Application Ser. No. 63/305,629 filed on Feb. 1, 2022; U.S. Provisional Application Ser. No. 63/310,071 filed on Feb. 14, 2022; U.S. Provisional Application Ser. No. 63/322,139 filed on Mar. 21, 2022; U.S. Provisional Application Ser. No. 63/330,725 filed on Apr. 13, 2022; U.S. Provisional Application Ser. No. 63/340,830 filed on May 11, 2022; and U.S. Provisional Application Ser. No. 63/351,751 filed on Jun. 13, 2022, each of which is incorporated herein by reference in its entirety for all purposes.
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.
| Number | Date | Country | |
|---|---|---|---|
| 63213642 | Jun 2021 | US | |
| 63222377 | Jul 2021 | US | |
| 63225284 | Jul 2021 | US | |
| 63231536 | Aug 2021 | US | |
| 63239866 | Sep 2021 | US | |
| 63239917 | Sep 2021 | US | |
| 63241952 | Sep 2021 | US | |
| 63245610 | Sep 2021 | US | |
| 63246754 | Sep 2021 | US | |
| 63255356 | Oct 2021 | US | |
| 63305629 | Feb 2022 | US | |
| 63310071 | Feb 2022 | US | |
| 63322139 | Mar 2022 | US | |
| 63330725 | Apr 2022 | US | |
| 63340830 | May 2022 | US | |
| 63351751 | Jun 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2022/034596 | Jun 2022 | WO |
| Child | 18393023 | US |
