Patent Grant
11371081
Not applicable.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 25, 2017, is named 112624_00851_SL.txt and is 217,680 bytes in size.
Synthetic biology is an emerging discipline that has great potential to respond to global pandemics. The increasing ability of synthetic biologists to repurpose and engineer natural biological components for practical applications has led to new opportunities for molecular diagnostics.
In the case of viral outbreaks, standard serological approaches such as antibody detection have limited diagnostic value due to cross-reactivity in patients that have previously been infected by other flaviviruses circulating in the region. As a result, accurate diagnosis requires nucleic acid-based detection methods, such as PCR and isothermal nucleic acid amplification. However, such techniques are expensive, require technical expertise to run and interpret, and use equipment that is incompatible with use in remote and low-resource locations where surveillance and containment are critically needed. Accordingly, there remains a need in the art for improved methods and devices for rapid detection of target nucleic acids, including pathogen-specific nucleic acids for infection detection and for accurate strain identification.
In a first aspect, provided herein is a method of detecting a target nucleic acid in a sample. In some cases, the method comprises or consists essentially of the steps of: (a) obtaining nucleic acid from a biological sample obtained from a subject; (b) amplifying the nucleic acid using isothermal amplification; (c) contacting the amplified nucleic acid to a toehold switch, wherein the toehold switch encodes a reporter protein and comprises one or more single-stranded toehold sequence domains that are complementary to a target nucleic acid or the reverse complement thereof, wherein the contacting occurs under conditions that allow translation of the coding domain in the presence of the target nucleic acid but not in the absence of the target nucleic acid, and detecting the reporter protein as an indicator that the target nucleic acid is present in the amplified nucleic acid of the subject; and (d) identifying the target nucleic acid as containing a target protospacer adjacent motif (PAM), wherein identifying comprises: (i) amplifying nucleic acid obtained from the biological sample using a reverse primer designed to append the trigger sequence of one or more toehold switch sequence domains; (ii) contacting the amplified nucleic acid of (i) to CRISPR/Cas under conditions that allow for sequence-specific cleavage of the contacted nucleic acid by CRISPR/Cas when the target PAM is present in the amplified nucleic acid; and (iii) detecting activation of the toehold switch, wherein activation does not occur in the event of CRISPR/Cas-mediated sequence-specific cleavage, thereby indicating the presence of the target PAM. The toehold switch can comprise one or more single-stranded toehold sequence domains, a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain comprising a ribosome binding site, and a coding domain. The toehold and stem domains can be complementary in sequence to a naturally occurring RNA. The loop domain can be complementary in sequence to a non-naturally occurring RNA. The target nucleic acid can be an RNA specific to a pathogen. The pathogen is selected from the group consisting of a virus, bacterium, fungus, and parasite. In some cases, the pathogen is a virus. The virus can be Zika virus. The virus can an American Zika variant (GenBank: KU312312). The virus strain can be an African Zika variant (GenBank: KF268950). The toehold switch can comprise an E. coli lacZ gene encoding β-galactosidase. Detecting activation of the one or more toehold switch sensors can comprise performing a LacZ-based colorimetric assay. Isothermal amplification can be selected from the group consisting of NASBA (nucleic acid sequence-based amplification), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and helicase-dependent amplification (HDA). The biological sample can be selected from the group consisting of blood, serum, urine, saliva, tissue, cell, and organ, or a fraction or portion thereof.
In another aspect, provided herein is a method of detecting a target nucleic acid in a sample. In some cases, the method comprises or consists essentially of: (a) obtaining RNA from a biological sample obtained from a subject; (b) amplifying the RNA using isothermal amplification; (c) contacting the amplified RNA to a toehold switch, wherein the toehold switch encodes a reporter protein and comprises one or more single-stranded toehold sequence domains that are complementary to a target RNA or the reverse complement thereof, wherein the contacting occurs under conditions that allow translation of the coding domain in the presence of the target RNA but not in the absence of the target RNA, and detecting the reporter protein as an indicator that the target RNA is present in the amplified RNA of the subject; and (d) identifying the target RNA as containing a target protospacer adjacent motif (PAM), wherein identifying comprises: (i) amplifying RNA obtained from the biological sample using a reverse primer designed to append the trigger sequence of one or more toehold switch sequence domains; (ii) contacting the amplified RNA of (i) to CRISPR/Cas under conditions that allow for sequence-specific cleavage of the contacted RNA by CRISPR/Cas when the target PAM is present in the amplified RNA; and (iii) detecting activation of the toehold switch, wherein activation does not occur in the event of CRISPR/Cas-mediated sequence-specific cleavage, thereby indicating the presence of the target nucleic acid. The toehold switch can comprise one or more single-stranded toehold sequence domains, a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain comprising a ribosome binding site, and a coding domain. The toehold and stem domains can be complementary in sequence to a naturally occurring RNA. The loop domain can be complementary in sequence to a non-naturally occurring RNA. The target nucleic acid can be an RNA specific to a pathogen. The pathogen can be selected from the group consisting of a virus, bacterium, fungus, and parasite. In some cases, the pathogen is a virus. The virus can be Zika virus. The virus can be an American Zika variant (GenBank: KU312312). The virus can be an African Zika variant (GenBank: KF268950). The toehold switch can comprise an E. coli lacZ gene encoding β-galactosidase. Detecting activation of the one or more toehold switch sensors can comprise performing a LacZ-based colorimetric assay. Isothermal amplification can be selected from the group consisting of NASBA (nucleic acid sequence-based amplification), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and helicase-dependent amplification (HDA). The biological sample is selected from the group consisting of blood, serum, urine, saliva, tissue, cell, and organ, or a fraction or portion thereof.
In a further aspect, provided herein is a method of detecting presence of virus in a sample. The method can comprise or consist essentially of the steps of: (a) obtaining RNA from a biological sample obtained from a subject; (b) amplifying the RNA using isothermal amplification; (c) contacting the amplified RNA to a toehold switch, wherein the toehold switch encodes a reporter protein and comprises one or more single-stranded toehold sequence domains that are complementary to an endogenous virus RNA sequence or the reverse complement thereof, wherein the contacting occurs under conditions that allow translation of the coding domain in the presence of the endogenous virus RNA but not in the absence of the endogenous virus RNA, and detecting the reporter protein as an indicator that the endogenous virus RNA is present in the amplified RNA of the subject. The virus can be Zika virus. The toehold switch can comprise one or more Zika genome-specific single-stranded toehold sequence domains, a thermodynamically stable double-stranded stem domain, a loop domain comprising a ribosome binding site, and a coding domain. The loop domain can be complementary in sequence to a naturally occurring RNA. The loop domain can be complementary in sequence to a non-naturally occurring RNA. The loop domain can be 11 nucleotides or 12 nucleotides. The toehold switch can comprise an E. coli lacZ gene encoding β-galactosidase. Isothermal amplification can be selected from the group consisting of NASBA (nucleic acid sequence-based amplification), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and helicase-dependent amplification (HDA).
In another aspect, provided herein is a device for identifying a pathogen, comprising a preserved paper test article, wherein a methods described herein is performed using the preserved paper test article. The paper test article can be preserved by freeze-drying.
In another aspect, provided herein is a kit for detecting a pathogen, comprising one or more of a device as described herein and an electronic optical reader.
In a further aspect, provided herein is a method of genotyping a nucleic acid molecule. The method can comprise or consist essentially of contacting the nucleic acid molecule with: a programmable nuclease; and a sgRNA, wherein the combination of the nuclease and sgRNA can specifically bind to at least one sequence variant of the nucleic acid molecule; and detecting the presence or absence of a cut in the nucleic acid molecule generated by the nuclease. In some cases, the method further comprises a first step of performing reverse transcription on a RNA molecule and performing 2nd strand DNA synthesis with a toehold primer to generate the nucleic acid molecule; and wherein the detecting step comprises: transcribing an RNA from the nucleic acid molecule after contacting it with the nuclease, using a primer which initiates transcription from a location distal of the sequence variation site with respect to the location of the toehold primer sequence; and contacting a sensor with the RNA resulting from step a) and detecting the presence or absence of sensor activation; wherein the sensor is activated if the nuclease is not able to cut the nucleic acid molecule in step a). The presence of a cut can indicate that the nucleic acid molecule has a sequence variant to which the sgRNA and nuclease can specifically bind. The presence of a cut can indicate that the nucleic acid molecule has a sequence variant to which the nuclease specifically binds. The programmable nuclease can be Cas. The sequence variant can occur at a PAM site. The nucleic acid molecule can be of human, animal, prokaryotic, eukaryotic, pathogenic, or synthetic origin. The nucleic acid molecule can be of viral origin. The viral nucleic acid molecule can be a Zika virus nucleic acid molecule. The sequence variant being detected can differentiate at least one of the African, American, and Asian Zika strains from the others. The sequence variant can be selected from Table 2. The sequence variant being detected can differentiate the African and American Zika virus strains. The sequence variant can be the SNP occurring at site 7330 of the African (GenBank: KF268950) and American (GenBank: KU312312) Zika strains. The sgRNA can have the sequence of SEQ ID NO: 1.
In another aspect, provided herein is a composition comprising a sgRNA which can specifically bind to a sequence flanking at least one sequence variant selected from Table 2, wherein the sequence variation occurs at a CRISPR/Cas PAM binding site. The sgRNA can comprise SEQ ID NO: 1. The sgRNA can be selected from Table 2.
In a further aspect, provided herein is a composition comprising a CRISPR/Cas nuclease and a sgRNA that specifically binds to a sequence flanking at least one sequence variant occurring in a population. The population can be a viral population. The viral population can be a Zika virus population. The variant can be selected from Table 2. The sgRNA can be selected from Table 2. The sgRNA can comprise SEQ ID NO: 1.
These and other features, objects, and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention.
The present invention will be better understood and features, aspects, and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:
While the present invention is susceptible to various modifications and alternative forms, exemplary embodiments thereof are shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description of exemplary embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though set forth in their entirety in the present application.
The methods and compositions provided herein are based at least in part on the Inventors' development of a diagnostic platform utilizing engineered biomolecular, nucleic acid-based sensors and CRISPR-based technology that permits rapid, specific, and low-cost detection of viral nucleic acids at clinically relevant concentrations. In particular, the inventors developed engineered biomolecular sensors for the specific detection of pathogen genomes such as viral RNA genomes.
Without being bound to any particular theory or mechanism of action, it is believed that the inventors addressed limitations in the practical deployment of nucleic acid-based molecular diagnostics by combining isothermal RNA amplification with toehold switch sensors on a freeze-dried, paper-based platform. By automating the amplification primer and sensor design process using in silico algorithms, the methods described herein provide clinically relevant sensitivity, discriminating between pathogen genotypes with single-base resolution.
Accordingly, in a first aspect, provided herein is a method of detecting a target nucleic acid in a biological sample obtained from a subject. As described herein, the method comprises or consists essentially of (a) obtaining nucleic acid (e.g., DNA, RNA) from a biological sample containing or suspected of containing a target nucleotide sequence; (b) amplifying the nucleic acid using a primer designed to hybridize to the target nucleotide sequence; (c) contacting the amplified nucleic acid to a toehold switch, where the riboregulator encodes a reporter protein and comprises one or more toehold sequence domains that are complementary to the target nucleotide sequence, where the contacting occurs under conditions that allow translation of the coding domain in the presence of the target nucleic acid but not in the absence of the target nucleic acid, and detecting the reporter protein as an indicator that the target nucleic acid is present in the amplified nucleic acid of the subject.
In certain embodiments, the target nucleotide sequence is a nucleic acid from a pathogen, where the biological sample contains or is suspected of containing the pathogen. Accordingly, the methods provided herein are useful to detect any pathogen or infectious agent. Pathogens and infectious agents may comprise viruses, (e.g., single stranded RNA viruses, single stranded DNA viruses, Zika virus, HIV, hepatitis A, B, and C virus, HSV, CMV EBV, HPV), parasites (e.g., protozoan and metazoan pathogens such as Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species), bacteria (e.g., Mycobacteria, in particular, M. tuberculosis, Salmonella, Streptococci, E. coli, Staphylococci), fungi (e.g., Candida species, Aspergillus species), Pneumocystis carinii, and prions. In certain embodiments, the pathogen is a virus, and the methods can be used to detect any virus. In other embodiments, the pathogens that are detected are bacteria, fungi, or parasites. An advantage of the methods and systems described herein is that they can be applied for the detection and identification of essentially any nucleic acid-containing organism. Accordingly, the pathogen or infectious agent can be virtually any pathogen or infectious agent for which genetic information (e.g., gene sequences) is available. In other cases, the target nucleic acid is human in origin. In such cases, the methods can be employed to detect one or more target nucleic acids in a biological sample such as a biological sample obtained for forensic analysis, for genotyping, and the like.
In such cases, the methods provided herein can further comprise identifying the pathogen detected in the biological sample. For example, the method can further comprise (i) amplifying RNA obtained from the biological sample; (ii) contacting the amplified RNA of (i) to a nuclease under conditions that allow for sequence-specific cleavage of the contacted RNA by the nuclease when a pathogen strain-specific protospacer adjacent motif (PAM) is present; and (iii) detecting activation of a toehold switch, where activation does not occur in the event of nuclease-mediated sequence-specific cleavage, thereby indicating the presence of the pathogen strain-specific PAM. In other cases, DNA is obtained from the biological sample and amplified as described above.
Other target nucleotide sequences include, without limitation, DNA or RNA sequences that can identify a species (e.g., ribosomal RNAs or DNAs); DNA or RNA sequences that are associated with a particular genetic condition (e.g., where the target comprises a single nucleotide polymorphism (SNP) for which PAM identification is advantageous, including, without limitation, BRCA1/BRCA2 mutations, cystic fibrosis, Duchenne muscular dystrophy, hemochromatosis); DNA or RNA sequences for identifying a particular person with high certainty (e.g., identifying a suspect in a criminal investigation; identifying a “high value target” in a military operation).
For forensic applications, the target nucleotide sequence can be a DNA or RNA sequence associated with one or more particular identifiable features (e.g., skin color, hair color, eye color). In such cases, a biological sample can be assayed to detect a target nucleic acid of an unknown subject or for comparison to samples from known individuals. For applications related to pathogen detection, detection of particular RNA sequences is advantageous for determining, for example, the life cycle stage of a pathogen associated with an infection. By way of example, particular target nucleic acids can be detected to detect the presence of malaria parasite Plasmodium falciparum and to determine whether the parasite is in a life cycle phase in which it can reproduce and, thus, transmit infection. Other applications for which the methods provided herein include, without limitation, profiling species in an environment (e.g., water); profiling species in an human or animal microbiome; food safety applications (e.g., detecting the presence of a pathogenic species, determining or confirming food source/origin such as type of animal or crop plant); obtaining patient expression profiles (e.g., detecting expression of a gene or panel of genes (e.g., biomarkers) to monitor the patient's response to a therapeutic regimen, to select a therapeutic regimen suitable for the patient, or to detect exposure of the patient to a toxin or environmental agent that affects expression of the gene or panel of genes; and molecular encryption applications such as marking certain products (e.g., high value products) using nucleic acid barcodes.
The nucleic acid molecule can be, e.g., an RNA, a DNA, an mRNA, and/or a genomic nucleic acid. In some embodiments of any of the aspects, the nucleic acid molecule can be human, animal, prokaryotic, eukaryotic, or pathogenic in origin. In some embodiments of any of the aspects, the nucleic acid molecule can be of viral origin. Nucleic acids and/or other moieties of the invention may be isolated. As used herein, “isolated” means separate from at least some of the components with which it is usually associated whether it is derived from a naturally occurring source or made synthetically, in whole or in part.
Nucleic acids and/or other moieties of the invention may be purified. As used herein, purified means separate from the majority of other compounds or entities. A compound or moiety may be partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
Biological samples appropriate for use according to the methods provided herein include, without limitation, blood, serum, urine, saliva, tissues, cells, and organs, or portions thereof.
Since the methods of the present invention provide single-base discrimination, the methods are particularly suited to distinguishing between genomes of a pathogen strain (e.g., to distinguish between pathogen strains) and/or identifying the presence of nucleic acids specific to a particular pathogen. As described herein, the methods incorporate isothermal RNA amplification and the sequence-specific nuclease activity of a CRISPR/Cas system. “Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas)” systems have been employed for targeted genome editing applications across many species. CRISPR systems belong to different classes, with different repeat patterns, sets of genes, and species ranges. The number of Cas genes at a given CRISPR locus can vary between species. The terms “Cas gene” and “CRISPR-associated (Cas) gene” are used interchangeably herein. A comprehensive review of the Cas protein family is presented in Haft et al. (2005) Computational Biology, PLoS Comput. Biol. 1:e60 (doi:10.1371/journal.pcbi.0010060). At least 41 CRISPR-associated (Cas) gene families have been described.
Without being bound to any particular theory or mechanism of action, Cas enzymes recognize a strain-specific protospacer adjacent motif (PAM) sequence. In one embodiment, in the case of the enzyme Cas9 the PAM sequence is NGG, where N can be any DNA base. Thus, a single base mutation, such as one that changes the sequence AGG to AAG, abolishes the PAM site and prevents Cas nuclease-based cleavage. As used herein, the term “protospacer” refers to the portion of a crRNA (or sgRNA) that is complementary to the genomic DNA target sequence. Generally, protospacers are usually 20 nucleotides in length. Referring to
As used herein, the term “toehold switch” generally refers to a nucleic acid-based regulator of gene expression, configured to repress or activate translation of an open reading frame and thus production of a protein. Toehold switches, which are a type of prokaryotic riboregulator, activate gene expression in response to cognate RNAs with essentially arbitrary sequences. Gene regulation is achieved through the presence of a regulatory nucleic acid element (the cis-repressive RNA or crRNA) within the 5′ untranslated region (5′ UTR) of an mRNA molecule. The cis-repressive nucleic acid element (crRNA) forms a hairpin structure comprising a stem domain and a loop domain through complementary base pairing. The hairpin structure blocks access to the mRNA transcript by the ribosome, thereby preventing translation. In some embodiments, the stem domain of the hairpin structure sequesters the ribosome binding site (RBS). In some embodiments, including for example embodiments involving eukaryotic cells, the stem domain of the hairpin structure is positioned upstream of the start (or initiation) codon, within the 5′ UTR of an mRNA. In some cases, riboregulators comprise synthetic (engineered) molecules. In other cases, toehold switches comprise endogenous, naturally occurring RNAs or regions thereof. See, for example, U.S. 2015/0275203. The stem domain can be as small as 12 bps, but in some cases will be longer than 12 bps, including 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs in length. In some cases, the loop domain is complementary to a naturally occurring RNA. In other cases, the loop domain is complementary to a non-naturally occurring RNA. The toehold domain can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides in length. Referring to
The toehold switch further comprises a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain comprising a ribosome binding site (RBS), and a coding domain. The unpaired region upstream of the RBS in a toehold switch can be shortened or lengthened to modulate protein output and, in turn, device dynamic range. In some cases, the toehold and stem domains are complementary in sequence to a naturally occurring RNA. In other cases, the sequence detected can also be the complement of the naturally occurring RNA. For example, after isothermal amplification, it is possible to transcribe the antisense of the RNA rather than the sense.
The toehold switch can further comprise a thermodynamically stable double-stranded stem domain, a loop domain comprising a ribosome binding site, and a coding domain. In some cases, the loop domain is complementary in sequence to a naturally occurring Zika virus RNA. In other cases, the loop domain is complementary in sequence to a non-naturally occurring RNA. Preferably, the loop domain is 11 nucleotides or 12 nucleotides in length. In some cases, the length of loop domains can be increased or decreased, for example, to alter reaction thermodynamics.
As shown in
Any isothermal amplification protocol can be used according to the methods provided herein. Exemplary types of isothermal amplification include, without limitation, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), signal mediated amplification of RNA technology (SMART), rolling circle amplification (RCA), isothermal multiple displacement amplification (IMDA), single primer isothermal amplification (SPIA), recombinase polymerase amplification (RPA), and polymerase spiral reaction (PSR available at nature.com/articles/srep12723 on the World Wide Web). In some cases, a forward primer is used to introduce a T7 promoter site into the resulting DNA template to enable transcription of amplified RNA products via T7 RNA polymerase. In other cases, a reverse primer is used to add a trigger sequence of a toehold sequence domain.
As used herein, “nuclease” refers to an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Nucleases can be site-specific, i.e. site-specific nucleases cleave DNA bonds only after specifically binding to a particular sequence. Therefore, nucleases specific for a given target can be readily selected by one of skill in the art. Nucleases often cleave both strands of dsDNA molecule within several bases of each other, resulting in a double-stranded break (DSB). Exemplary nucleases include, but are not limited to Cas9; Cas13; meganucleases; TALENs; zinc finger nucleases; FokI cleavage domain; RNA-guided engineered nucleases; Cas-derived nucleases; homing endonucleases (e.g. I-AniI, I-CreI, and I-SceI) and the like. In some embodiments of any of the aspects, the nuclease is an endonuclease. As used herein, “endonuclease” refers to an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids within a polynucleotide, e.g., cleaving a phosphodiester bond that is not either the 5′ or 3′ most bond present in the polynucleotide. In other embodiments of any of the aspects, the nuclease is a meganuclease. As used herein, “meganuclease” refers to endonucleases, which have a large recognition sequence (e.g., dsDNA sequences of 12-40 bp). Due to the size of the recognition sequences, meganucleases are particularly specific. Meganuclease specificity can be engineered. In some embodiments of any of the aspects, the meganuclease can be a LAGLIDADG homing endonuclease (SEQ ID NO: 2).
In some embodiments, the nuclease can be an engineered nuclease. As used herein, the terms “engineered” and “genetically engineered” are used interchangeably and refer to the aspect of having been manipulated by the hand of man. For example, a nuclease is considered to be “engineered” when the sequence of the nuclease is manipulated by the hand of man to differ from the sequence of the nuclease as it exists in nature. As is common practice and is understood by those in the art, progeny and copies of an engineered polynucleotide and/or polypeptide are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity. Methods of engineering nucleases to achieve a desired sequence specificity are known in the art and are described, e.g., in Kim and Kim. Nature Reviews Genetics 2014 15:321-334; Kim et al. Genome Res. 2012 22:1327-1333; Belhaj et al. Plant Methods 2013 9:39; Urnov et al. Nat Rev Genet 2010 11:636-646; Bogdanove et al. Science 2011 333:1843-6; Jinek et al. Science 2012 337:816-821; Silva et al. Curr Gene Ther 2011 11:11-27; Ran et al. Cell 2013 154:1380-9; Carlson et al. PNAS 212 109:17382-7, Guerts et al. Science 2009 325:433-3; Takasu et al. Insect Biochem Mol Biol 2010 40:759-765; and Watanabe et al. Nat. Commun. 2012 3; each of which is incorporated by reference herein in its entirety.
In some embodiments, the nuclease is a programmable nuclease. As used herein “programmable nuclease” refers to a nuclease that has been engineered to create a double-stranded break (DSB) or nick at a nucleic acid sequence that the native nuclease would not act upon, e.g. the sequence specificity of the nuclease has been altered. As described herein, programmable nucleases can be used to genotype a nucleic acid and/or determine the sequence of a nucleic acid. In particular, programmable nucleases can differentiate between point mutations or SNPs, e.g., SNPs that occur in a PAM site. In one aspect of any of the embodiments, described herein is a method of genotyping a nucleic acid molecule, the method comprising: a) contacting the nucleic acid molecule with: a programmable nuclease; and a single guide (“sgRNA”) which can specifically bind to at least one sequence variant of the nucleic acid molecule; and b) detecting the presence or absence of a cut in the nucleic acid molecule generated by the nuclease. In one aspect of any of the embodiments, described herein is a method of genotyping a nucleic acid molecule, the method comprising: a) contacting the nucleic acid molecule with: a programmable nuclease; and a sgRNA wherein the combination of the nuclease and sgRNA can specifically bind to at least one sequence variant of the nucleic acid molecule; and b) detecting the presence or absence of a cut in the nucleic acid molecule generated by the nuclease. In some embodiments of any of the aspects, the presence of a cut indicates that the nucleic acid molecule has a sequence variant for which the sgRNA is specific. In some embodiments of any of the aspects, the presence of a cut indicates that the nucleic acid molecule has a sequence variant to which the nuclease specifically binds. In some embodiments of any of the aspects, the presence of a cut indicates that the nucleic acid molecule has a sequence variant for which the sgRNA is specific and has a sequence variant to which the nuclease specifically binds. In some embodiments of any of the aspects, the presence of a cut indicates that the nucleic acid molecule has a sequence variant to which the sgRNA and nuclease can specifically bind.
By way of non-limiting example, the programmable nuclease can be Cas9; Cas13, a Cas nickase mutant; TALEN; ZFNs; Cpf1; and/or SaCas9. In some embodiments of any of the aspects, the programmable nuclease is Cas9. In some embodiments, the programmable nuclease is Cas9. In some embodiments of any of the aspects, the programmable nuclease is S. pyogenes Cas9 or a variant thereof, e.g., New England Biolabs #M0386 (Ipswich, Mass.). When Cas9 nuclease (or Cas9-derived nuclease) is selected for use, the nuclease will generate a cut and/or nick where the guide RNA hybridizes to the nucleic acid molecule.
In order for a Cas nuclease to recognize and cleave a target nucleic acid molecule, a CRISPR targeting RNA (“crRNA”) and trans-activating crRNA (“tracrRNA”) must be present. crRNAs hybridize with tracrRNA to form a hybrid guide RNA (“gRNA”) which then associates with the Cas9 nuclease. Alternatively, the gRNA can be provided as a single contiguous RNA, and forms a single guide RNA (“sgRNA”). Once the sgRNA is complexed with Cas, the complex can bind to a target nucleic acid molecule. The sgRNA binds specifically to a complementary target sequence via a target-specific sequence in the crRNA portion (e.g., the spacer sequence), while Cas itself binds to a protospacer adjacent motif (CRISPR/Cas protospacer-adjacent motif; PAM). The Cas nuclease then mediates cleavage of the target nucleic acid to create a double-stranded break within the sequence bound by the sgRNA. Different Cas enzymes have different PAM recognition sequences. For example, S. pyogenes Cas9 requires a NGG PAM sequence while other CRISPR/Cas systems have been described in other prokaryotic species, which recognize a different PAM sequence (e.g., CCN, TCN, TTC, AWG, CC, NNAGNN, NGG, NGGNG).
In some embodiments of any of the aspects, the sgRNA is provided as a single continuous nucleic acid molecule. In some embodiments of any of the aspects, a hybrid gRNA is provided as a set of hybridized molecules, e.g., a crRNA and tracrRNA.
In embodiments in which the nuclease to which the amplified DNA or RNA is contacted is a Cas nuclease, a method of detecting a viral nucleic acid comprises or consists essentially of: (a) obtaining DNA or RNA from a biological sample obtained from a subject; (b) amplifying the DNA or RNA using a primer designed to append a trigger sequence of one or more toehold sequence domains; (c) contacting the amplified DNA or RNA to a toehold switch, where the riboregulator encodes a reporter protein and comprises one or more toehold sequence domains, where the contacting occurs under conditions that allow translation of the coding domain in the presence of the endogenous virus DNA or RNA but not in the absence of the endogenous virus DNA or RNA, and detecting the reporter protein as an indicator that the endogenous virus DNA or RNA is present in the amplified DNA or RNA of the subject; and (d) identifying the strain of virus, where identifying comprises: (i) amplifying DNA or RNA from the biological sample; (ii) contacting the amplified DNA or RNA of (i) to Cas (e.g., Cas9, Cas13) under conditions that allow for sequence-specific cleavage of the contacted RNA by Cas (e.g., Cas9, Cas13) when a virus strain-specific protospacer adjacent motif (PAM) is present; and (iii) detecting activation of the toehold switch, where activation does not occur in the event of Cas-mediated sequence-specific cleavage, thereby indicating the presence of the virus strain-specific PAM. For example, the methods provided herein can be used to distinguish between viral strains, e.g., where one strain comprises a PAM site while the second strain comprises a SNP that eliminates the PAM site, such an American Zika variant (GenBank: KU312312) and an African Zika variant (GenBank: KF268950), and also between other flavivirus strains. See,
In some cases, the one or more toehold sequence domains are complementary to an endogenous virus DNA or RNA sequence. In such cases, where the toehold switch recognizes an endogenous RNA sequence, there is no requirement for a primer that appends a toehold sequence domain.
With respect to the amplification step, the target sequence for a toehold switch is in some cases added via an amplification primer for the NASBACC process. In other cases, a toehold switch that detects an endogenous pathogen DNA or RNA sequence is used.
In another aspect, provided herein is a method of detecting Zika virus in a sample. The methods can comprises, or consist essentially of, (a) obtaining RNA from a biological sample obtained from a subject; (b) amplifying the RNA using isothermal amplification; and (c) contacting the amplified RNA to a riboregulator, wherein the riboregulator encodes a reporter protein and comprises one or more toehold domains that is complementary to a Zika virus RNA, wherein the contacting occurs under conditions that allow translation of the coding domain in the presence of the Zika virus RNA but not in the absence of the Zika virus RNA, and detecting the reporter protein as an indicator that the Zika virus RNA is present in the amplified RNA of the subject.
In some cases, it may be advantageous to adapt the methods described herein for high-throughput, reproducible, and rapid detection, for example in a clinical setting. When riboregulator output is coupled to a reporter element, such as a LacZ reporter element, the riboregulator acts as a genetically encodable sensor and detectable probe for endogenous DNA or RNA (e.g., endogenous pathogen DNA, endogenous pathogen RNA) in a sample. For example, such toehold switches can be provided in a device configured for rapid, reproducible detection in a clinical setting. In some cases, the device comprises a preserved paper test article, upon which any step(s) of the method provided herein can be performed. In preferred embodiments, the paper test article is preserved by freeze-drying. The reporter element can be a reporter protein, e.g., a polypeptide with an easily assayed enzymatic activity or detectable signal that is naturally absent from the host cell. Exemplary but non-limiting reporter proteins include lacZ, catalase, xy1E, GFP, RFP, YFP, CFP, neomycin phosphotransferase, luciferase, mCherry, and derivatives or variants thereof. In some embodiments of any of the aspects, the reporter protein is suitable for use in a colorimetric assay. Examples of genes encoding fluorescent proteins that may be used in accordance with the invention include, without limitation, those proteins provided in U.S. Patent Application No. 2012/0003630 (see Table 59 therein), incorporated herein by reference.
In some cases, the device is used with a portable electronic reader. In this manner, the electronic reader serves as companion technology that provides robust and quantitative measurements of device outputs. As shown in
As used herein, “sequence variations” can refer to substitutions, insertions, deletions, duplications, and/or rearrangements. Sequence variations of a locus occurring in a population are referred to as alleles. Sequence variations can be present in (and therefore, detected in) the gDNA and/or mRNA of a gene. In some embodiments of any of the aspects, the sequence variation is a point mutation, e.g. a single nucleotide polymorphism (SNP). As used herein, a “point mutation” refers to the identity of the nucleotide present at a site of a mutation in the mutant copy of a genomic locus (including insertions and deletions), i.e., it refers to an alteration in the sequence of a nucleotide at a single base position from the wild type sequence. A SNP (single nucleotide polymorphism) is one type of point mutation that occurs at the same genomic locus between different individual subjects or entities in a population or different strains in a species. SNPs can be allelic. At least four alleles of a SNP locus are possible, although SNPs that vary only between two nucleotides at the target site are not uncommon.
In some embodiments of any of the aspects, the target nucleic acid is a Zika virus nucleic acid molecule, e.g., a Zika virus genomic molecule or a molecule transcribed from the Zika virus genome.
The methods described herein can permit identification of the species of virus present in a sample (e.g., a sample obtained from a subject), and/or permit identification of the strain of a virus present in a sample based upon sequence variations found between species and/or strains. Such information can be used to direct treatment, e.g., different strains of Zika virus are known to cause different symptoms and secondary conditions at varying frequencies. In some embodiments of any of the aspects, the sequence variant being detected differentiates at least one of the African, American, and Asian Zika strains from the others. Exemplary sequence variants that differentiate these strains are provided in Table 5.
In certain embodiments, provided herein is a method for genotyping a nucleic acid molecule. The method can comprise or consist essentially of contacting the nucleic acid molecule with a programmable nuclease and a sgRNA, where the combination of the nuclease and sgRNA can specifically bind to at least one sequence variant of the nucleic acid molecule; and detecting the presence or absence of a cut in the nucleic acid molecule generated by the nuclease. In some cases, the method further comprises a first step of performing reverse transcription on a RNA molecule and performing 2nd strand DNA synthesis with a toehold primer to generate the nucleic acid molecule. In such cases, the detecting step comprises: (i) transcribing an RNA from the nucleic acid molecule after contacting it with the nuclease, using a primer which initiates transcription from a location distal of the sequence variation site with respect to the location of the toehold primer sequence; and (ii) contacting a sensor with the RNA resulting from step (a) and detecting the presence or absence of sensor activation; wherein the sensor is activated if the nuclease is not able to cut the nucleic acid molecule in step (a). As used herein, the term “toehold primer” refers to an oligonucleotide primer configured to add a detectable tag or label sequence, where the tag or label sequence is detectable by a downstream nucleic acid sensor.
Primers and sgRNAs can readily be designed for a given variant according to the principles described herein. Cas9 selectively cleaves DNA only in the presence of an NGG protospacer adjacent motif (PAM). As demonstrated herein, e.g. in Example 1, numerous strain-specific PAM sites exist. The reverse transcription primer is designed to specifically bind near the selected PAM site such that reverse transcription proceeds towards the PAM site. The sgRNA and/or guide RNA is then designed to specifically bind to a sequence located between the PAM site and the sequence to which the reverse transcription primer specifically binds. Tools for designing primers and sgRNAs are known in the art. For example, a primer sequence can be selected to have a desired TM (melting temperature) using any of a number of widely available algorithms (e.g., OLIGO™ (Molecular Biology Insights Inc. Colorado) primer design software and VENTRO NTI™ (Invitrogen, Inc. California) primer design software and programs available on the internet, including Primer3 and Oligo Calculator). Algorithms are also widely available for sgRNA design (e.g., several online tools (e.g., The Broad Institute's sgRNA Design tool, CRISPR Design or CHOPCHOP, which are available on the internet). Methods of making primers and other nucleic acid sequences (e.g., oligonucleotides, sgRNAs) are well known in the art, and numerous commercial sources offer oligonucleotide synthesis services suitable for providing molecules according to the methods and compositions described herein, e.g. INVITROGEN™ Custom DNA Oligos; Life Technologies; Grand Island, N.Y. or custom DNA Oligos from IDT; Coralville, Iowa).
In some embodiments of any of the aspects, the sequence variant being detected differentiates the African and American Zika virus strains. In some embodiments of any of the aspects, the sequence variant is the SNP occurring at site 7330 of the African (GenBank: KF268950) and American (GenBank: KU312312) Zika strains. In some embodiments of any of the aspects, the sgRNA has the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the method differentiates the African and American Zika virus strains by detecting the presence or absence of the SNP occurring at site 7330 of the African (GenBank: KF268950) and American (GenBank: KU312312) Zika strains, and the sgRNA has the sequence of SEQ ID NO:1.
Articles of Manufacture
In another aspect, the present invention provides articles of manufacture useful for detecting a virus or identifying a virus strain. In preferred embodiments, the article of manufacture is a kit for detecting a virus, where the kit comprises a plurality of preserved paper test articles and an electronic optical reader. Optionally, a kit can further include instructions for performing the virus detection and/or strain identification methods provided herein.
In some aspects of any of the embodiments, described herein is a composition comprising a Cas nuclease and a sgRNA which can specifically bind to at least one sequence variant occurring in a population. In some aspects of any of the embodiments, described herein is a composition comprising a Cas9 nuclease and a sgRNA which can specifically bind to at least one sequence variant occurring in a population, wherein the sequence variation occurs at the Cas9 PAM binding site.
In some aspects of any of the embodiments, described herein is a composition comprising a Cas nuclease and a sgRNA which can specifically bind to at least one sequence variant occurring in a viral population. In some aspects of any of the embodiments, described herein is a composition comprising a Cas9 nuclease and a sgRNA which can specifically bind to at least one sequence variant occurring in a viral population, wherein the sequence variation occurs at the Cas9 PAM binding site.
In some aspects of any of the embodiments, described herein is a composition comprising a Cas nuclease and a sgRNA which can specifically bind to at least one sequence variant occurring in a Zika virus population. In some aspects of any of the embodiments, described herein is a composition comprising a Cas9 nuclease and a sgRNA which can specifically bind to at least one sequence variant occurring in a Zika virus population, wherein the sequence variation occurs at the Cas9 PAM binding site.
In some aspects of any of the embodiments, described herein is a composition comprising a Cas nuclease and a sgRNA which can specifically bind to at least one sequence variant selected from Table 10. In some aspects of any of the embodiments, described herein is a composition comprising a Cas9 nuclease and a sgRNA which can specifically bind to at least one sequence variant selected from Table 5, wherein the sequence variation occurs at the Cas9 PAM binding site.
In some aspects of any of the embodiments, described herein is a composition comprising a Cas9 nuclease and a sgRNA comprising SEQ ID NO:1. In some aspects of any of the embodiments, described herein is a composition comprising a Cas9 nuclease and a sgRNA comprising SEQ ID NO:1, wherein the sequence variation occurs at the Cas9 PAM binding site. In some embodiments of any of the aspects, the sgRNA consists of SEQ ID NO:1.
In some aspects of any of the embodiments, described herein is a sgRNA which can specifically bind to a sequence flanking at least one sequence variant selected from Table 5, wherein the sequence variation occurs at a Cas9 PAM binding site. In some aspects of any of the embodiments, described herein is a composition a sgRNA comprising SEQ ID NO:1. In some aspects of any of the embodiments, described herein is a composition a sgRNA consisting of SEQ ID NO:1.
Methods for sgRNA selection and design are described elsewhere herein. In some embodiments of any of the aspects, a sgRNA which can specifically bind to a sequence flanking a given sequence variant can comprise a 20 nt sequence complementary to a sequence found from 1-30 nucleotides from the sequence variation. In some embodiments of any of the aspects, a sgRNA which can specifically bind to a sequence flanking a given sequence variant can comprise a 20 nt sequence complementary to a sequence found from 1-25 nucleotides from the sequence variation. In some embodiments of any of the aspects, a sgRNA which can specifically bind to a sequence flanking a given sequence variant can comprise a 20 nt sequence complementary to a sequence found from 1-20 nucleotides from the sequence variation.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein, “about” means within 5% of a stated concentration range or within 5% of a stated time frame.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
Having now described the invention, the same will be illustrated with reference to certain examples, which are included herein for illustration purposes only, and which are not intended to be limiting of the invention.
Reference is now made to the following examples, which together with the above descriptions illustrate the invention in a non-limiting fashion.
Materials and Methods
In Silico Sensor Design and DNA Synthesis:
A set of 48 toehold switch sensors and corresponding NASBA primers were generated using an integrated in silico design algorithm.
DNA Sensor Assembly:
Toehold switch constructs were amplified from DNA templates (Integrated DNA Technologies) and ligated to the lacZ reporter gene via PCR. Plasmids were constructed for characterization of the top six toehold switches (
Cell-Free Reactions:
Details of RNA sensor validation are described in Pardee et al. (2014). Briefly, amplified sensor DNA was column purified and tested on paper discs (2 mm) containing freeze-dried, cell-free reactions (NEB, PURExpress) in the presence or absence of trigger RNA coding for a complementary region of the Zika virus genome (128-178 nts). The cell-free reactions consisted of: NEB Solution A (40%) and B (30%), chlorophenol red-b-D-galactopyranoside (Sigma, 0.6 mg/ml), RNase inhibitor (Roche, 03335402001; 0.5%), and linear DNA constructs encoding the toehold sensors (0.33 nM). The paper discs (Whatman, 1442-042) were blocked in 5% BSA overnight prior to use. Trigger RNA was produced using T7 RNAP-based transcription (Epicenter ASF3257) from linear DNA templates. Paper-based reactions (1.8 μl) were incubated at 37° C. using either our companion electronic reader inside a humidified chamber or a plate reader (BioTek Neo). For the in-house reader, paper discs were placed into 2 mm holes in a removable acrylic chip; for the plate reader, paper discs were placed into black, clear bottom 384-well plates (Corning 3544).
NASBA:
For NASBA reactions, the trigger elements (128-178 nts) were extended by 100 nts on the 5′ and 3′ ends with the relevant Zika genome sequence to provide suitable template RNAs. RNA amplicons were spiked into 7% human serum (Sigma H4522) where indicated. Reaction Buffer (Life Sciences NECB-24; 33.5%), Nucleotide Mix (Life Sciences NECN-24; 16.5%), RNase inhibitor (Roche, 03335402001; 0.5%), 12.5 mM of each NASBA primer (2%), nuclease free water (2.5%), and RNA amplicon (20%) were assembled at 4° C. and incubated at 65° C. for 2 min, followed by a 10 min incubation at 41° C. Enzyme Mix (Life Sciences NEC-1-24; 25%) was then added to the reaction (for a final volume of 5 and the mixture was incubated at 41° C. for 2 hr. unless noted otherwise. For output reads with paper-based toeholds, the NASBA reactions were diluted 1:7 in water. See Table 2 for primer sequences.
Lentivirus Preparation and Processing:
HEK293FT cells (Life Technologies, R70007) used for virus packaging were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 4 mM GlutaMAX (ThermoFisher Scientific). 12 hr. prior to transfection, 6.5×106 cells were seeded in a 10 cm dish. 7.5 mg psPAX2, 2.5 mg pMD2.G, and 10 mg pSB700 modified to include a Zika or Dengue RNA fragment were transfected using the HeBS—CaCl2 method. Media was changed 12 hr. post-transfection. 27 hr. after changing media, viral supernatant was harvested and filtered using a 0.45 mm syringe filter. Viral supernatant was then purified with ViraBind Lentivirus Purification Kit (Cell Biolabs VPK-104) and buffer exchanged into 1×PBS with Lenti-X Concentrator (Clontech, 631231). Viral RNA concentration was quantified using QuickTiter Lentivirus Quantification Kit (VPK-112). Virus samples were spiked into 7% human serum at a final volume of 25 μl. Samples were heated to 95° C. for 1 and 2 min and used as input to NASBA.
Zika Virus Preparation and Processing:
100 μl of Zika virus isolate (MR 766) was utilized for infection of 106 Vero cells in 4 ml of media (DMEM supplemented with 2% fetal calf serum [FCS] and penicillin-streptomycin). The supernatant was removed after 2 hr. of incubation at 37° C. and replaced with fresh media (DMEM, 10% FCS) for 48 hr. of infection. Cell debris was removed by centrifugation at 1,500 rcf for 10 min, and aliquots of the virus were stored at −80° C. until use. The virus was buffer exchanged into 1×PBS with Lenti-X Concentrator (Clontech, 631231). Viral RNA concentrations were determined from virus purified with the QIAamp Viral RNA Mini Kit (QIAGEN 52904) and confirmed with qRT-PCR. The titer of the Zika virus used was 6.7×107 infectious units per milliliter (Lambeth et al., 2005). Virus samples were spiked into 7% human serum at a final volume of 30 μl. Samples were heated to 95° C. for 2 min and used as input to NASBA. NASBA primers were re-designed to accommodate the MR 766 strain sequence.
Dengue Orthogonality:
Genomic RNA from three Dengue serotypes was purified using the QIAamp Viral RNA Mini Kit (QIAGEN 52904). Dengue 1 (GenBank: KM204119), Dengue 2 (GenBank: KM204118), Dengue 4 (GenBank: AF326573). NASBA reactions using the sensor 32B primer set were performed on 30 pM RNA for 2 hr. NASBA reactions were diluted 1:7 in water and used to rehydrate freeze-dried, paper-based reactions containing sensor 32B.
Electric Optical Reader:
The portable device consists of four layers housed within a laser-cut acrylic box fastened together with metal screws and mounting brackets (
Calculation of Fold Change:
The calculation of fold change for plate reader data was done by first subtracting the background absorbance measured from paper-based reactions that did not contain sensor DNA or trigger RNA. These normalized values were smoothed to reduce measurement noise using a three-point average of the time point and the data collected 10 min before and after. The minimum value of each well was then adjusted to zero. For data presented in
NASBA-CRISPR Cleavage (NASBACC):
Reactions were performed in a 5 μl volume containing (NASBA buffer), 1 μl of a 250 nM Cas9 nuclease (NEB, M0386), and 250 nM purified gRNA (GeneArt precision gRNA synthesis kit, ThermoFisher Scientific, A29377) mix, 3 nM NASBACC primers, and 0.4 units of RNase inhibitor (NEB, M0314). The forward NASBACC primer is composed of the reverse complement of the trigger H sequence (5′-GTT TGA ATG AAT TGT AGG CTT GTT ATA GTT ATG TTT-3′ (SEQ ID NO: 3)) and the forward binding sequence of the (region 32) NASBA primers. The reverse NASBACC primer contains the T7 promoter sequence (5′-CTA ATA CGA CTC ACT ATA GG-3′ (SEQ ID NO: 4)) followed by the reverse binding sequence of the (region 32) NASBA primers. The assembled reaction was incubated at 37° C. for 2 to 6 hours. For toehold activation assay on freeze-dried paper, NASBACC reactions were diluted 1:10 in nuclease-free water.
Zika Virus Stock Production for Macaque Infection:
ZIKV strain H/PF/2013 (GenBank accession number: KJ776791), originally isolated from a 51-year-old female in France returning from French Polynesia with a single round of amplification on Vero cells, was obtained from Xavier de Lamballerie (European Virus Archive, Marseille France). Virus stocks were prepared by inoculation onto a confluent monolayer of C6/36 mosquito cells. A single harvest of virus with a titer of 1.26×106 PFU/ml for the Asian-lineage (equivalent to 1.43×109 vRNA copies/ml) was used.
Viremic Plasma Processing:
All Indian-origin rhesus macaque monkeys from which plasma was isolated were cared for by the staff at the Wisconsin National Primate Research Center (WNPRC) in accordance with the regulations and guidelines outlined in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals and the recommendations of the Weatherall report. This study was approved by the University of Wisconsin-Madison Graduate School Institutional Animal Care and Use Committee (Animal Care and Use Protocol Number G005401). For all procedures (i.e., physical examination, virus inoculation, blood and swab collection), animals were anesthetized with an intramuscular dose of ketamine (10 ml/kg). Blood samples were obtained using a vacutainer system or needle and syringe from the femoral or saphenous vein. For processing, plasma was diluted 1:10 in nuclease free water, heated to 95° C. for 2 min, and immediately added to a NASBA reaction. NASBA was run for 3 hr.
Zika Virus Challenge of Macaques, Plasma Collection, and Processing:
The virus stock was thawed, diluted in PBS to the appropriate concentration for each challenge, and loaded into a 1 ml syringe that was kept on ice until challenge. Animals were anesthetized as described above, and 1 ml of inocula was administered subcutaneously over the cranial dorsum. At the conclusion of the procedure, animals were closely monitored by veterinary and animal care staff for adverse reactions and signs of disease. Fresh plasma and PBMC were isolated from EDTA-treated whole blood by Ficoll density centrifugation at 1860 rcf for 30 min. The plasma layer was collected and centrifuged for an additional 8 min at 670 rcf to remove residual cells. The supernatant plasma was then filtered over a 0.45 μm syringe filter. Collected plasma was diluted 1:10 in nuclease free water. Diluted samples were heated to 95° C. for two minutes and immediately added to a NASBA reaction as described above. NASBA was run for three hours.
qRT-PCR to Determine Macaque Plasma Viral Loads:
Viral RNA was extracted from 300 μl of plasma using the Viral Total Nucleic Acid Purification Kit (Promega) on a Maxwell 16 MDx instrument. Viral RNA was quantified by qRT-PCR using the primers and probe designed by Lanciotti et al. (2008). The RT-PCR was performed using the SuperScript III Platinum one-step quantitative RT-PCR system (Invitrogen) on the LightCycler 480 instrument (Roche Diagnostics). Primers and probe were used at final concentrations of 600 nm and 100 nm, respectively, along with 150 ng random primers (Promega). Cycling conditions were as follows: 37° C. for 15 min, 50° C. for 30 min, and 95° C. for 2 min, followed by 50 cycles of 95° C. for 15 seconds and 60° C. for 1 min. Virus concentration was determined by interpolation onto an internal standard curve composed of seven 10-fold serial dilutions of a synthetic ZIKV RNA fragment based on the Asian lineage.
Results
In Silico Toehold Switch Design:
Toehold switch sensors are programmable synthetic riboregulators that control the translation of a gene via the binding of a trans-acting trigger RNA. The switches contain a hairpin structure that blocks gene translation in cis by sequestration of the ribosome binding site (RBS) and start codon. Upon a switch binding to a complementary trigger RNA, sequestration of the RBS and start codon is relieved, activating gene translation (
Toehold switch sensors for sequence-based detection of Zika virus were generated using an expanded version of the previously developed in silico design algorithm (Green et al., 2014). The modified algorithm screened the genome of the Zika strain prevalent in the Americas (Genbank:KU312312) for regions compatible with RNA amplification and toehold switch activation. The selected Zika genome regions were then computationally filtered to eliminate potential homology to the human transcriptome and to a panel of related viruses, including Dengue and Chikungunya. A total of 24 unique regions of the Zika genome compatible with downstream sensing efforts were identified.
Two toehold switches, each utilizing a different design scheme, were designed for each region, resulting in a total of 48 sensors. The first design scheme, termed the A series, utilizes a modification to the original toehold switch (Green et al., 2014) that reduces the size of the loop domain from 18 nts to 11 nts (
Rapid In Vitro Sensor Assembly and Screening:
In vitro assembly and initial screening of all 48 sensors took place in a 7 hr. time period, with low costs associated with sensor development (DNA input $20 USD/sensor) and testing ($0.10-$1/test). All 48 sensors and 24 targeted genomic regions were assembled in-house using in vitro protocols. Toehold switches were constructed by ligating the sensors (˜130 nt) to a LacZ reporter element in a single 2 hr. PCR-based step. Sensor performance screening to assess each sensor against its respective trigger RNA element (Zika genome fragment) was completed using low volume, cell-free transcription and translation reactions on paper. We found that 25 (52%) of the 48 sensors produce a fold change of five or greater in the presence of the appropriate trigger element (128-178 nucleotide regions of the Zika genome;
Assessing and Improving Zika Virus Sensor Sensitivity:
We selected top performing sensors from both the A and B series for trigger RNA titration experiments and found that all chosen sensors were activated with as little as 30 nM of trigger RNA (
Though the sensors displayed specificity for their respective Zika RNA trigger, they were unable to detect clinically relevant RNA concentrations. Zika viral loads have been documented as high as 202×106 copies/ml (365 fM) in urine (Gourinat et al., 2015). However, viral loads in saliva and serum are reportedly even lower, with 3×106 copies/ml (4.9 fM) (Barzon et al., 2016) documented in patient saliva and 2.5×106 copies/ml (4.1 fM) (Zika Experimental Science Team, 2016) and 7.2×105 copies/ml (1.2 fM) (Lanciotti et al., 2008) in primate and patient serum, respectively. Accordingly, to increase the sensitivity of our diagnostic platform, we incorporated an isothermal RNA amplification technique known as NASBA (nucleic acid sequence-based amplification) into our workflow (
NASBA is a promising candidate for use with our diagnostic scheme because it is known to be extremely sensitive and has a proven track record in field-based diagnostic applications (Cordray and Richards-Kortum, 2012). The amplification process begins with reverse transcription of a target RNA that is mediated by a sequence-specific reverse primer to create an RNA/DNA duplex. RNase H then degrades the RNA template, allowing a forward primer containing the T7 promoter to bind and initiate elongation of the complementary strand, generating a double-stranded DNA product. T7-mediated transcription of the DNA template then creates copies of the target RNA sequence. Importantly, each new target RNA can be detected by the toehold switch sensors and also serve as starting material for further amplification cycles. NASBA requires an initial heating step (65° C.), followed by isothermal amplification at 41° C. (
NASBA was performed on trigger RNA corresponding to Zika genomic regions for sensors 27B and 32B. Trigger RNAs were spiked into either water or human serum (7%) to more closely mimic clinical samples. NASBA reactions were run for 2 hr. and then applied to freeze-dried, paper-based sensors. We saw detection with Zika sensors from NASBA reactions initiated with as little as 3 fM of trigger RNA (
Field-Ready Diagnostic Platform:
To move our experiments toward conditions more representative of those found in clinics worldwide, we focused on three key efforts: (1) testing sensor specificity against related viruses that share clinical symptoms, partial homology, and geographic range with Zika virus; (2) building a second-generation portable, battery-powered reader to provide lab-quality results in low resource environments; and (3) developing a low-cost and tractable method for viral RNA extraction.
Although our sensor design algorithm screened for Zika genomic sequences that are mostly distinct from those of related viruses, the targeted Zika sequences do share substantial similarity (51%-59%) with their Dengue virus counterparts (
As part of our efforts to advance the paper-based sensor platform toward field-ready diagnostics, we designed a second generation portable electronic reader to serve as an accessible, low-cost companion technology that provides robust and quantitative measurements of sensor outputs. The electronic reader was assembled using readily available consumer components, open-source code, and laser-cut acrylic housing, with a total cost of just under $250 (
To demonstrate the utility of the companion reader, we monitored detection of 1 fM and 3 fM of Zika RNA amplicons that had been amplified in NASBA reactions for 2.5 hr. The reader detected significant signal from both samples, which are within the reported range of Zika virus in patient serum (1.2 fM) and urine (365 fM) (Gourinat et al., 2015; Lanciotti et al., 2008), after just over 20 min (
Our next challenge was to develop a technique to release RNA from the viral capsid using simple methodology compatible with low-resource environments. To this end, we tested the efficacy of boiling viral samples to break down the capsid. For initial development, we engineered lentivirus, which is also an RNA virus, to encapsulate the regions of either the Zika or Dengue genomes that correspond to the sensor 32B target sequence (
NASBA-CRISPR Cleavage Assay to Discriminate Between Zika Strains:
During epidemic outbreaks, it is often valuable to monitor pathogen lineage and geographic spread. In some cases, genetic variants may be responsible for different clinical manifestations of infection. For example, the Zika strain found in Brazil has been uniquely connected with higher incidences of fetal microcephaly and Guillain-Barré syndrome (Calvet et al., 2016; Mlakar et al., 2016). To allow for strain-specific detection and tracking, we developed an assay that provides single-base discrimination in a manner that is compatible with our freeze-dried sensor platform. Our assay, which we term NASBA-CRISPR Cleavage (NASBACC), leverages the sequence-specific nuclease activity of CRISPR/Cas9 to discriminate between viral lineages (
Using the paper-based system, sensor 32B was able to distinguish between Zika and Dengue RNA sequences. However, this sensor could not discriminate between the African (GenBank: KF268950) and American (GenBank: KU312312) Zika variants (
Diagnostic Workflow Validation with Active Zika Virus:
We next sought to validate our sensor platform with live Zika virus. First, we verified that our amplification and detection scheme could successfully detect full-length genomic RNA purified from Zika virus (Uganda strain MR 766) (
Once we confirmed that the sensors behaved as expected on full-length genomic RNA, we sought to validate the sample preparation scheme and diagnostic workflow from start to finish. Active Zika virus was cultured in the laboratory and spiked into human serum (7%) at a final concentration of 10 fM, to mimic a clinical sample. The viral sample was then heated to 95° C. for 2 min, and the resulting lysate was subjected to NASBA amplification for three hours. Sensor activation from the NASBA-amplified viral sample was monitored on the portable electronic reader. We successfully detected activation of sensor 32B from a diagnostic workflow initiated with live Zika virus (
For the final validation of our system, we acquired and tested plasma samples from a viremic macaque infected with Zika virus (GenBank: KJ776791) (Zika Experimental Science Team, 2016). The macaque was found to have a plasma viral load of 1.7×106 copies/ml (2.8 fM) by a standard qRT-PCR protocol, which was within the detection limits of our platform as tested on synthetic RNA amplicons (
In Silico Strategy for Toehold Switch Sensor and NASBA Primer Design.
An integrated in silico strategy was developed for generating optimal NASBA primers and toehold switches for detection of Zika. Mirroring the procedure used for running the paper-based diagnostic assay, a set of optimal primers was initially generated for the NASBA reaction and then a series of toehold switch designs screened for activity on the RNA transcripts produced by NASBA was developed.
Identification of Optimal NASBA Primers for Zika Amplification.
A set of potential primer pairs with favorable characteristics for NASBA reactions as described by Deiman et al. (Deiman et al., 2002) was generated. The Zika genome was analyzed to identify all potential forward and reverse priming sites that had the following characteristics:
All subsequences from the Zika genome that did not satisfy requirements 1 to 4 were immediately eliminated from consideration. The remaining primers were then analyzed for characteristics 5-8 and the deviation from optimal 50% GC content, with each parameter converted into a numerical score. The forward NASBA primers, which append the T7 promoter site to NASBA DNA intermediate, were modeled with this T7 promoter site present. Accordingly, the prefix sequence AATTCTAATACGACTCACTATAGGGAGAAGG (SEQ ID NO: 5) (T7 promoter sequence underlined) was appended to the 5′ end of each forward primer. The resulting scores were combined and used to estimate the overall favorability of every potential primer in the Zika genome. Following this initial screen, the top 2% of all the potential primers were then compared to the rest of the Zika genome to determine the longest contiguous region of the primer that matched more than one site in the genome. This comparison provided a coarse check of primer specificity in advance of more detailed primer specificity screening conducted later in the design process. The length of this contiguous region for each primer was then incorporated into a final score for each primer.
The first stage of screening resulted in a set of forward and reverse primers to provide optimal characteristics for binding to the target genome; however, it did not consider other important effects on NASBA efficiency, namely the length and secondary structure of the amplicon produced by NASBA. To consider these amplicon-related effects, the binding sites for each primer on the genome were identified and, if more than one primer acted at the same site, the primer awarded the highest favorability score was selected. A set of potential forward and reverse primer pairs was constructed based on the recommended amplicon lengths for successful NASBA reactions. Although NASBA is known to work best with amplicons having lengths between approximately 120-nts to 250-nts (Deiman et al., 2002), this rapid screening approach initially employs synthetic DNA strands as templates for transcribing the target RNA. Since the length of these DNA oligos (IDT Ultramers) is currently limited to 200-nts including the T7 promoter site, primer pairs were instead examined for amplicons ranging from 120- to 176-nts in length. After applying all the above constraints on primer and amplicon sequences, a set of 4351 potential NASBA primer pairs remained. The RNA amplicons generated for each of these primers pairs were then assessed for their secondary structure. NASBA reactions are known to be more effective when applied to templates having low secondary structure. Consequently, the degree of amplicon single-strandedness was examined using the NUPACK ensemble defect function (Zadeh et al., 2011a, 2011b).
Next, each of the potential NASBA primers were then coarsely screened for sequence similarities with 11 viruses known to be closely related to Zika: Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4, West Nile virus, St. Louis encephalitis virus, yellow fever virus, Powassan virus, Semliki Forest virus, O'nyong'nyong virus, and Chikungunya virus. Sequence similarities were estimated by determining the maximum contiguous sequence in the primer that was found in any of the 11 related viruses. More stringent specificity screening was carried out in later stages as described below.
After all the above screening procedures, the final NASBA primer pairs were sorted by quality after taking into account the favorability scores of each primer, the secondary structure of the amplicon, and the potential for non-specific binding with other viruses.
Identification of Optimal Toehold Switches for NASBA Products.
Out of the 4351 NASBA amplicons, the top 1025 were selected as potential targets for toehold switches. The in silico design process for these toehold switches followed closely that previously used for designing mRNA sensors in vivo (Green et al., 2014) and on paper (Pardee et al., 2014). Briefly, toehold switch mRNA sensors were designed that hybridized to the NASBA amplicon at 1-nt increments. This sliding window encompassed the internal region of the NASBA product outside of the primer binding sites. This internal region was selected to avoid any potential for sensor activation or competitive binding by residual NASBA primers. The resulting toehold switches were analyzed for secondary structure and toehold availability, and screened to eliminate any sensors with unwanted in-frame stop codons in the output gene sequence. The target transcript itself was again assayed for single-strandedness and availability of the sensor binding sites. The above factors were incorporated into a sensor design score as described previously (Green et al., 2014). The highest scoring toehold switch sensor for each amplicon was then passed on to the final selection stage.
Final Design Selection Process.
After the above screening and design stages, the set of 1025 NASBA primer pairs was assembled with corresponding optimized toehold switch sensors. The top overall designs were selected by combining the favorability scores obtained from the NASBA and toehold switch evaluation steps. The primer and sensor sequences from these top designs were then tested for specificity against the human transcriptome and the same panel of closely related viral genomes listed above using NCBI/Primer-BLAST. Moreover, they were screened for specificity within the Zika genome itself. Ultimately, the top 24 designs that survived the Primer-BLAST specificity stage were selected for testing using our rapid in vitro screening approach.
Modifying the Toehold Switch Sensor Design for Decreased Signal Leakage.
Detailed studies of the toehold switch design parameters (Green et al., 2014) and thermodynamic considerations suggested two simple strategies for decreasing leakage from the toehold switches: reducing the size of the loop containing the ribosome binding site (RBS) in the sensor, and further stabilization of the sensor stem. Both these strategies were applied in the Zika-specific toehold switches.
The ON and OFF state signals from the toehold switches increase as the size of the loop in the switch RNA increases. This effect is likely due to two factors: increased accessibility of the RBS to the ribosome, which promotes translation in the presence or absence of the target RNA; and entropic effects that discourage stem formation as the loop becomes longer. Conversely, decreasing the size of the loop is associated with lower leakage, albeit with a decrease in ON state activity. Stabilizing the switch RNA stem by adding additional base pairs or by eliminating the downstream refolding domain (Green et al., 2014) increases the free energy required to unwind the sensor stem and thus encourages decreased signal leakage.
In accordance with above factors, two different types of toehold switches were tested aiming to lower leakage. The first type of sensor, referred to as the A series, are nearly identical to those previously used for mRNA sensing (Green et al., 2014; Pardee et al., 2014), except their loop domain has been reduced from 18-nts to 11-nts. These A sensors retained the downstream refolding domain to encourage sensor triggering and they all have the same sequence at the top of the sensor stem-loop (GUUAUAGUUAUGAACAGAGGAGACAUAACAUGAAC (SEQ ID NO: 6)) as illustrated in
The second type of sensor, referred to as the B series, possesses a stem that has been lengthened by one base pair overall and a loop region that is only 12-nts. Importantly, the B sensors also lack the downstream refolding domain to further stabilize the OFF state. The parental toehold switch for the B sensors exhibited extremely low leakage in preliminary measurements in paper-based reactions and provided a sizeable ON/OFF ratio of ˜600-fold regulating GFP expression in E. coli. These B sensors all featured the same conserved sequence (GGACUUUAGAACAGAGGAGAUAAAGAUG (SEQ ID NO: 7)) at the top of their stem-loops as illustrated in
Considerations of Sequence Information in Design of the Biomolecular Diagnostics
Evolutionary drift. At the time we began our experiments, few complete genomes for the Zika virus had been reported. In fact, the first complete genome of the strain circulating in the Americas was only published on January 7 in The Lancet (Enfissi et al., 2016). However, previous comparisons of Zika strains do provide information on the degree of evolutionary drift for the virus. Haddow et al. found that a Zika strain isolated in Malaysia in 1966 differed by only 4.3% in nucleotide sequence from isolates obtained Micronesia and Cambodia in 2007 and 2010, respectively (Haddow et al., 2012). Authors also found ≤11.7% nucleotide sequence difference between African and Asian virus lineages, which they argued provided sufficient conserved sequence for genetic tests for both lineages. More recent studies have shown the rate of mutation of Zika is ˜10-3 nucleotide changes/site/year, which is relatively high among flaviviruses, but a manageable rate for our diagnostic assay (Faria et al., 2016).
Specificity of the NASBA/toehold switch isothermal assay. Since the experiments are performed at mostly 41° C. and 37° C., melting-temperature-dependent tuning of primer specificity is not possible in our assays. The benefit of this temperature limitation is that our NASBA and toehold switch detection schemes are able to tolerate mismatches and compensate for variability in the sequence of the target RNA molecules. The binding between the toehold switch 32B, our highest performing sensor, and RNAs from homologous regions in Zika strains isolated from Africa (Uganda, Nigeria, Senegal) and Asia (Malaysia, Cambodia, French Polynesia) were analyzed. All these strains are predicted to fully activate the toehold sensors even with up to 4-nt (11%) mismatches.
Compensating for evolutionary drift. The above analysis is borne out in data showing that sensor 32B can detect the target sequence from both the American and African strains of the virus (
Integration with signature erosion analysis tools. Software such as BioVelocity (Sozhamannan et al., 2015) and TOPSI (Vijaya Satya et al., 2010) are adept at determining conserved sequence regions across multiple genomes and eliminating those shared with other pathogens or humans. Using these valuable tools, a set of specific sites for NASBA priming and toehold switch binding can be generated and then subjected to the same screening by toehold secondary structure and NASBA primer characteristics described in this work.
Freeze-Dried NASBA.
For freeze-dried NASBA experiments, Enzyme Mix was lyophilized separately from the other components. The solution containing reaction buffer, nucleotide mix, RNase inhibitor, and primers was reconstituted in 15% DMSO, while the Enzyme Mix was reconstituted in nuclease-free water.
Freeze-Dried CRISPR/Cas9 Nuclease Assay.
Reactions were performed in a 30 j·tl volume of 1×Cas9 Nuclease Reaction Buffer (NEB #M03865) containing 30 nM of guide RNA (gRNA), 30 nM of Cas9 Nuclease (S. pyogenes, NEB #M03865), and 3 nM of substrate DNA (pAG_TS1_KS001 plasmid). Six gRNA sequences targeting the lacZ gene (Doench et al., 2014) inserted into plasmid pAG_TS1_KS001 were used for the CRISPR/Cas9 freeze-dried assay. All components except substrate DNA were first combined in a 27 μl reaction volume and incubated for 10 minutes at 25° C. to allow Cas9+gRNA to form duplexes. For fresh reactions, 3 μl of a 30 nM solution of substrate DNA was added to the solution. For freeze-dried reactions, the 27 μl solution was lyophilized overnight and reconstituted with 30 j·tl of a solution containing 3 nM of substrate DNA. After the addition of substrate DNA, the solution was incubated for 1 h at 37 C and run on a 1% agarose gel for fragment analysis.
Care and Use of Macaques at the Wisconsin National Primate Research Center.
All Indian-origin rhesus macaque monkeys from which plasma was isolated were cared for by the staff at the Wisconsin National Primate Research Center (WNPRC) in accordance with the regulations and guidelines outlined in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals and the recommendations of the Weatherall report. This study was approved by the University of Wisconsin-Madison Graduate School Institutional Animal Care and Use Committee (Animal Care and Use Protocol Number G005401). For all procedures (i.e., physical examination, virus inoculation, blood and swab collection), animals were anesthetized with an intramuscular dose of ketamine (10 ml/kg). Blood samples were obtained using a vacutainer system or needle and syringe from the femoral or saphenous vein.
Zika Virus Stock Production for Macaque Infection.
ZIKV strain H/PF/2013 (GenBank accession number: KJ776791), originally isolated from a 51-year-old female in France returning from French Polynesia with a single round of amplification on Vero cells, was obtained from Xavier de Lamballerie (European Virus Archive, Marseille France). Virus stocks were prepared by inoculation onto a confluent monolayer of C6/36 mosquito cells. A single harvest of virus with a titer of 1.26×106 PFU/ml for the Asian-lineage (equivalent to 1.43×109 vRNA copies/ml) was used.
Zika Virus Challenge of Macaques, Plasma Collection and Processing.
The virus stock was thawed, diluted in PBS to the appropriate concentration for each challenge, and loaded into a 1 ml syringe that was kept on ice until challenge. Animals were anesthetized as described above, and 1 ml of inocula was administered subcutaneously over the cranial dorsum. At the conclusion of the procedure, animals were closely monitored by veterinary and animal care staff for adverse reactions and signs of disease. Fresh plasma and PBMC were isolated from EDTA-treated whole blood by Ficoll density centrifugation at 1860 rcf for 30 min. The plasma layer was collected and centrifuged for an additional 8 min at 670 rcf to remove residual cells. The supernatant plasma was then filtered over a 0.45 μm syringe filter. Collected plasma was diluted 1:10 in nuclease free water. Diluted samples were heated to 95 C for two minutes and immediately added to a NASBA reaction as described above. NASBA was run for three hours.
Cas9 Interferes with T7-Mediated RNA Production.
RNA can be produced in vitro from a DNA template containing the T7 promoter sequence (5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO: 768)). If the DNA template has viable CRISPR-Cas9 target sites, addition of Cas9+gRNA complex to the in vitro RNA production reaction leads to the production of truncated RNA product.
In
Cas9 can be Freeze-Dried and Remain Active.
It was next tested whether guide RNA targeting the LacZ sequence contained in the LacZ gene would remain active after being freeze-dried. In particular, a reaction containing Cas9+gRNA complex in Cas9 buffer (NEB #m0386) was freeze dried and re-hydrated with a solution containing 3 nM of template DNA. The template DNA is a supercoiled plasmid containing the LacZ gene.
In
Cas9 without Guide RNA does not Interfere with NASBA.
The presence of Cas9 nuclease alone does not interfere with NASBA reactions. The non-reactivity of NASBA reactions to Cas9 nucleases was demonstrated by adding Cas9 nuclease and Cas9 buffer to a NASBA reaction. Since the stability of thermal Cas9 has not been demonstrated above 37° C., the NASBA reaction was performed at 37° C.
Cas9 with a gRNA Targeting a Site Lacking a PAM Site does not Interfere with NASBA.
A key aspect of this invention is that a single base mutation affecting a PAM site will present Cas9 from binding/cleaving template DNA. To demonstrate this, a guide RNA with the 20-bp spacer sequence neg4-noGG (neg4-noGG TTTCAAGAATGGAAAACATC (SEQ ID NO: 771)) was designed to be homologous a region of the American strain (GenBank: KU312312) of Zika RNA, but at a location that lacks a PAM site (ATG): neg4-noNGG, Loc=2621 of GenBank: KU312312, sequence:
For comparison, another guide RNA sequence with the spacer sequence pos4 (pos4: GATCTCCTCTGTTTCAAGAA (SEQ ID NO: 773)) was designed near a PAM site (TGG) to cut the template DNA and interfere with NASBA amplification: pos4, Loc=2610 of GenBank: KU312312, sequence:
Cas9 with a gRNA that Targets Reverse-Transcribed RNA Interferes with NASBA.
This is demonstrated in
Cas9 can Interfere with NASBA Amplification when Using Low Reverse Transcription (RT) Primers Concentration of 3 nM or Less.
The amount of template DNA that is generated during the reverse-transcription step of a NASBA reaction has to remain low. Using the conditions detailed above herein (250 nM final concentration of Cas9+gRNA), it was found that the amount of reverse transcription primer present in the NASBA has to be 3 nM or lower in order to efficiently cleave any intermediate template DNA generated during a NASBA reaction. A final concentration of 1 nM is preferable.
Table Showing the PAM Site Creation/Annihilation Following a Random Mutation (Table 4).
This table shows all the possible outcomes of a random mutation, where a PAM site can be created or annihilated.
Algorithm to Detect Diverging PAM Sites Between Two Strains.
List of PAM sites that differ between the American-African strains (572,
TGGAGTCCCGCTGCTAATGA
GTTTTAGAGCTAGAAATAGCAAGTTAAAAT
AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT.
The present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.
This application represents the national stage entry of PCT International Application No. PCT/US2017/034545, filed on May 25, 2017, and claims the benefit of U.S. Provisional Patent Application No. 62/341,221, filed on May 25, 2016, and U.S. Provisional Patent Application No. 62/403,778, filed on Oct. 4, 2016, each of which is incorporated by reference it its entirety as if fully set forth herein.
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| 20150275203 | Green | Oct 2015 | A1 |
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| 20190218624 | Green | Jul 2019 | A1 |
| 20190256898 | Green | Aug 2019 | A1 |
| 20190276901 | Green | Sep 2019 | A1 |
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