WO2021216573A1 - Système évolutif, facile à déployer et réactifs associés pour la détection à base de nucléase associée à crispr de matériel génétique pathogène - Google Patents

Système évolutif, facile à déployer et réactifs associés pour la détection à base de nucléase associée à crispr de matériel génétique pathogène Download PDF

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WO2021216573A1
WO2021216573A1 PCT/US2021/028192 US2021028192W WO2021216573A1 WO 2021216573 A1 WO2021216573 A1 WO 2021216573A1 US 2021028192 W US2021028192 W US 2021028192W WO 2021216573 A1 WO2021216573 A1 WO 2021216573A1
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rna
sequence
dna
detection
crispr
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PCT/US2021/028192
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English (en)
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Carolina ARIAS GONZALEZ
Diego ACOSTA-ALVEAR
Max Wilson
Kenneth KOSIK
Michael Costello
Jose Carlos PONCE ROJAS
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The Regents Of The University Of California
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Publication of WO2021216573A1 publication Critical patent/WO2021216573A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present invention relates to diagnostic, prognostic and therapeutic methods, systems and kits. Specifically, the invention relates to systems and methods of detecting various pathogens, including, without limitation, SARS-CoV-2.
  • COVID-19 pandemic presents the world with an unprecedented public health challenge.
  • the lack of COVID-19 symptoms in a significant proportion (estimates range from 18 to 29%) of SARS-CoV-2 -infected individuals fuels covert transmission of the virus. Even in cases in which symptoms do present, the virus can be transmitted to others before symptom onset.
  • a hurdle to deploying massively widespread, recurrent testing is the availability of reagents and specialized equipment. Suggested solutions to make sample collection scalable include laboratory-made Viral Transport Media and 3D-printed swabs and bypassing biochemical RNA extraction through heat and chemical extraction techniques. Yet, there have been few end-to-end solutions to SARS-CoV-2 testing that fulfill the requirements of being immediately scalable and low-cost, without sacrificing sensitivity. Here, we focused on developing a CRISPR-based SARS-CoV-2 RNA detection method that is low cost, highly sensitive, and easy to deploy at sites with minimal infrastructure.
  • Casl2 and Casl3 (CRISPR-based) methods have been transformative with regards to pathogen detection. Yet, because of the global demand for testing, key reagents in these protocols are difficult to obtain. Challenges remain as to using a pathogen detection system in a field deployable manner.
  • some aspects of the invention provide for a system for pathogen detection that affords easy reagent accessibility, scalability, and is amenable to field deployment, which is also sensitive and has a high fidelity.
  • Methods for detecting a level and/or existence of a predefined nucleic acid sequence in a subject include assaying a biological sample obtained from a subject desiring a determination regarding a disease, condition and/or pathogenic infection, and detecting the level and/or existence of the predefined nucleic acid sequence, wherein the assaying step comprises preparing nucleic acid from the biological sample, amplifying the nucleic acid to generate copies of a template sequence containing a promoter sequence recognizable by a polymerase, and incubating the copies of the template sequence with a CRISPR-associated nuclease, a guide RNA, a polymerase that recognizes the promoter sequence, rNTP or dNTP, and a reporter molecule.
  • Various embodiments provide that the methods of detection, when combined with a miniPCR for amplification and a P51 molecular fluorescence viewer, allows for detection of a target SARS-CoV-2 sequence within about 2 hours for a sensitivity of at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, or preferably at least 97%), or detecting at least 10 copies of the template sequence per microliter, and a specificity of at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or preferably at least 98%).
  • the methods for detection includes preparing nucleic acid from a biological sample using a new lysis formulation.
  • compositions, or formulations, for cell lysis and thereby extraction of nucleic acid from a biological sample comprise sodium acetate, linear polyacrylamide, octylphenoxypolyethoxyethanol (IGEPAL CA-630), 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), and water, in a pH ranging from 7.0 to 7.4.
  • IGEPAL CA-630 octylphenoxypolyethoxyethanol
  • HEPES 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethanesulfonic acid
  • water in a pH ranging from 7.0 to 7.4.
  • the extraction buffer comprises tris(2- carboxyethyl)phosphine (TCEP) and glycerol.
  • TCEP tris(2- carboxyethyl)phosphine
  • the extraction buffer consists essentially of TCEP and glycerol.
  • the extraction buffer consists of TCEP, glycerol, and water.
  • the extraction buffer comprises TCEP and glycerol, further comprises dithiothreitol (DTT), polyethylene glycol, or both.
  • the extraction buffer comprises TCEP and glycerol, and does not comprise DTT and polyethylene glycol.
  • the DTT if present, is at about 2 mM, and PEG8000, if present, is at about 10% (w/v).
  • compositions or formulations comprise sodium acetate at about 450 mM, the linear polyacrylamide at about 50 pg/mL, the IGEPAL CA-630 at about 0.5% (v/v), and the HEPES-KOH at about 20 mM, in an aqueous medium so that the formulation has a pH of about 7.2, and when present, the glycerol at about 20% (v/v) (or about 10% v/v), the TCEP at about 20 mM.
  • a system comprising one or more, or all of: a lysis solution to prepare nucleic acid from a biological sample, a miniPCR thermocycler and Taq DNA polymerase to generate copies of a template sequence containing a linear amplification promoter from the prepared nucleic acid, a CRISPR-associated nuclease, a guide RNA, a reporter molecule, and a polymerase for linear amplification of the template sequence, and a P51 molecular fluorescence viewer or a quantitative fluorescence detection equipment, or a lateral flow assay test strip.
  • FIG. 1 is a schematic flow chart of Casl3-based detection methods and CREST modifications (i-iii) Standard sample collection, RNA extraction, and reverse transcription (iv) Amplification using cost-effective Taq polymerase and DIY-Bio thermocyclers instead of isothermal reactions (v) Transcription and Casl3 activation are followed by fluorescence detection of de-quenched poly-U cleavage reporter visualized with blue LED ( ⁇ 495nm) and orange filter or other fluorescence detection system.
  • FIG.2 depicts detection of SARS-CoV-2 RNA using CREST. Fluorescence visualization of Nl, N2, and N3 synthetic targets using P51 visualizer.
  • FIG. 3A and 3B depict comparative analysis of method sensitivity and cost per test.
  • FIG. 4A-4C depict purification of Casl3a.
  • ST column strep-tactin column.
  • FIG. 5A-5H depict optimization of Cast 3a detection parameters.
  • the total volume of Casl3a detection reaction does not impact signal sensitivity
  • 5B Casl3a detection under various rNTP final concentrations (for each target, bars as shown for 10 mM, 5 mM, 0.5 mM, and 0 mM of rNTP from left to right).
  • 5D Casl3a detection with various final concentrations of murine RNase Inhibitor (RI).
  • 5G Casl3a detections with various cleavage reporter concentrations (for each target, bars as shown from left to right are for 2000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM of cleavage reporter, respectively).
  • FIG. 5I-5K depict limit of detection determination for CREST.
  • (51) Coarse and (5 J) fine concentration scan of negative nasal -pharyngeal swabs spiked with heat inactivated virus at indicated concentrations before RNA was extracted and analyzed via CREST. Each concentration was extracted in three independent biological replicates shown.
  • FIG. 6A-6F depict detection sensitivity for N2 and N3 targets.
  • (6C & 6F) TaqMan RT-qPCR for N2 and N3 targets. Experiments were performed in duplicates or in triplicates (error bars SD). Statistical significance was determined using one-way ANOVA with Dunnetf s method (relative to water negative control).
  • FIG. 6G depicts Casl3 detection master mix shows no loss in sensitivity of SARS-Cov-2 Nl upon multiple freeze-thaw cycles.
  • FIG. 61 depicts side-by-side comparison of CREST and RT- qPCR done on 95 de-identified oropharyngeal samples taken from asymptomatic individuals for Nl, N2 and RNaseP with positive and negative control samples.
  • FIG. 8 depicts a comparison of TaqMan cycle thresholds obtained by the Qiagen QiAmp solid-phase RNA extraction and an exemplary PEARL solution in Example 2.1., in connection with an embodiment of the invention. Lower threshold values represent a more efficient nucleic acid extraction.
  • FIG. 9A is a PEARL workflow.
  • FIG. 9B is comparative RT-qPCR analysis of the levels of SARS-CoV-2 nucleocapsid (Nl) and RNaseP RNA sequences in de-identified SARS-CoV-2 positive and negative clinical specimen samples after RNA extraction using PEARL or an RNA extraction kit (QIAamp Mini Elute Virus Spin Kit) in nasopharyngeal swab samples. Dotted lines indicate a limit of detection of 36 cycles. We obtained Cq values for Nl below our limit of detection in 9 negative samples out of 67. No Cq values were obtained for Nl in the remaining 58 samples. Data points correspond to the reciprocal of the Cq value (1/Cq), which is directly proportional to input RNA. P value: Spearman’s correlation coefficient (p).
  • FIG. 10A and 10B depict RT-qPCR analysis of the levels of KSHV (LANA, GFP) and host b-actin (ACTB) mRNAs (10A), and those of their corresponding genomic sequences (10B).
  • FIG. IOC is Western blot analysis of the expression of KSHV (LANA, GFP) and host (HSP70) proteins. Inf, infected; Uninf, uninfected.
  • FIG. 11A depicts RT-qPCR analysis of the levels of ZIKV non- structural proteins NS1 and NS5 and host b-actin (ACTB) mRNAs.
  • FIG. 11B depicts qPCR analysis of the expression host ACTB genomic DNA sequences in ZIKV-infected samples.
  • FIG. IOC depicts western blot analysis of expression of ZIKV (NS2B) and host (HSP70) proteins. *non-specific band.
  • FIG. 12 depicts threshold values obtained by RT-qPCR for SARS-CoV-2 N1 and N2 viral transcripts, and the RNase P host mRNA, obtained using PEARL extracts. Different volumes of swab samples with the indicated sample to PEARL-lysis-buffer ratios were used to determine the conditions that maximize readout.
  • FIG. 13A-13C depict assessment of the integrity of PEARL-extracted analytes. RNA, DNA, and proteins were extracted from 1 x 10 6 HeLa cell using PEARL to their integrity was assessed by electrophoresis analyses.
  • FIG. 13A depicts Total RNA isolated from PEARL extracts using TRIzol reagent. The RNA was incubated with RNase A for 15 min at 37 °C or left intact, separated on a urea- PAGE gel, and stained with SYBR Safe. *partial digestion of rRNAs in the sample. Major RNA species are indicated.
  • FIG. 13B depicts PEARL extracted total DNA after RNA and protein digestion.
  • FIG. 13C depicts PEARL protein extracts separated by SDS-PAGE and stained with Coomassie blue after nuclease digestion.
  • FIG. 14A depicts dot blot immunodetection of ZIKV (NS2B) and host (HSP70) proteins.
  • FIG. 14B depicts dot blot immunodetection of KSHV (GFP, LANA) and host (HSP70) proteins. Inf, infected; Uninf, uninfected.
  • FIG. 15A in the top left panel depicts 3D render of a hand-powered centrifuge. CAD files can be found 3dprint.nih.gov/discover/3dpx-014683.
  • FIG. 15A in the top right panel depicts assembled handpowered centrifuge with its rotor loaded and the driving strings supercoiled.
  • FIG. 15A in the bottom left panel depicts actuation of the hand-powered centrifuge by string supercoiling.
  • FIG. 15A in the bottom right is close-up image of the spinning rotor during actuation.
  • FIG. 15A in the top left panel depicts 3D render of a hand-powered centrifuge. CAD files can be found 3dprint.nih.gov/discover/3dpx-014683.
  • FIG. 15A in the top right panel depicts assembled handpowered centrifuge with its rotor loaded and the driving strings supercoiled.
  • FIG. 15A in the bottom left panel depicts actuation of the hand-powered centrifuge by string supercoil
  • FIG. 15B depicts Cq values obtained by probing for host and KSHV mRNAs using RT-qPCR using PEARL extracts prepared with either a benchtop laboratory centrifuge or our hand-powered centrifuge, “Handfuge”.
  • FIG. 15C depicts dot blots obtained by probing for host and KSHV proteins in PEARL extracts prepared as in FIG. 15B.
  • FIG. 16 depicts a genomic map of some SARS-CoV-2 genome regions detected in Examples 3 and 1.
  • FIG. 17A, 17B depict viral loads in asymptomatic and confirmed positive individuals.
  • (17B) Median, solid line.
  • NS: p 0.63 for RNase P Kruskal-Wallis test.
  • predefined nucleic acid sequence refers to a polynucleotide with a known sequence, or a selected, targeted, or of-interest polynucleotide whose presence or level in a sample is to be determined but whose sequence is known or with whom a polynucleotide sequence is commonly and/or specifically associated by one skilled in the art.
  • phrase “recognition” as used herein with respect to interactions between two polynucleotide molecules refers to complementary base-pairing between a molecule that recognizes and a molecule being recognized.
  • the CRISPR-associated nuclease is a RNA-targeting effector protein.
  • the RNA-targeting effector protein is Casl3a, or formerly known as C2c2.
  • the Casl3a is from an organism of a genus selected from the group consisting of: Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifr actor, Mycoplasma, Campylobacter , and Lachnospira
  • the C2c2 effector protein is an organism selected from the group consisting of: Leptotrichia, Listeria, Corynebacter,
  • guide RNA is also called guide CRISPR RNA (crRNA).
  • the guide RNA is designed to detect protospacer adjacent motif (PAM) or the equivalent for a target RNA or DNA.
  • the protospacer adjacent motif (PAM) or PAM-like motif directs binding of the CRISPR-associated nuclease/crRNA complex as disclosed herein to the target locus of interest.
  • PAM may be used interchangeably with the term “PFS” or “protospacer flanking site” or “protospacer flanking sequence”.
  • the one or more guide RNAs are designed to detect a single nucleotide polymorphism, splice variant of a transcript, or a frameshift mutation in a target RNA or DNA. In further embodiments, the one or more guide RNAs are designed to bind to one or more target molecules that are diagnostic for a disease state.
  • target sequence refers to a polynucleotide sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence may comprise RNA polynucleotides.
  • target RNA refers to an RNA polynucleotide being or comprising the target sequence.
  • the target RNA may be a RNA polynucleotide or a part of a RNA polynucleotide to which a part of the gRNA, i.e.
  • target sequence comprises a protospacer adjacent motif site, or a single nucleotide polymorphism, a splice variant or a frameshift mutation, of the predefined nucleic acid sequence.
  • a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. In various embodiment, the subject is a human in the methods.
  • disease, condition, or a (pathogenic) infection refers to an infection, an organ disease, a blood disease, an immune system disease, a cancer, a brain and nervous system disease, an endocrine disease, a pregnancy or childbirth-related disease, an inherited disease, or an environmentally-acquired disease.
  • the disease state is cancer or an autoimmune disease or an infection.
  • the infection is caused by a virus, a bacterium, or a fungus, or the infection is a viral infection.
  • the viral infection is caused by a double-stranded RNA virus, a positive sense single-stranded RNA virus, a negative sense single-stranded RNA virus, a retrovirus, a double-stranded DNA virus, a single-stranded DNA virus, or a combination thereof, or the viral infection is caused by a Coronaviridae virus, a Picomaviridae virus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus, a Bornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, or a Deltavirus, or the viral
  • the phrase “biological sample” may be a blood sample or mucous sample.
  • the biological sample refers to mucous samples that may be obtained from nasopharyngeal swab, oropharyngeal swab/sampling, nasal swab, or buccal swab.
  • the term “specifically binding,” or “selectively binding,” refers to the interaction between binding pairs. In some embodiments, the interaction has an affinity constant of at most KG 6 moles/liter, at most KG 7 moles/liter, or at most KG 8 moles/liter. In other embodiments, the phrase “specifically binds” or “selectively binding” refers to the specific binding of one protein to another protein or ligand, wherein the level of binding is statistically significantly higher than the background control for the assay.
  • CREST Casl3-based, Rugged, Equitable, Scalable Testing.
  • CREST addresses two of the main hurdles — reagent accessibility and cost — that limit the scalability of Cas 13 -based testing, by taking advantage of low-cost, easy-to-use thermocyclers and fluorescent visualizers.
  • RT-qPCR reverse transcription quantitative polymerase chain reaction
  • a high-level general workflow of the methods of detection disclosed herein includes distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system comprising a CRISPR-associated nuclease, one or more guide RNAs, a reporter molecule; incubating the sample or set of samples under conditions sufficient to allow binding of the one or more guide RNAs to one or more target molecules; activating the CRISPR-associated nuclease via binding of the one or more guide RNAs to the one or more target molecules, wherein activating the CRISPR-associated nuclease results in modification of the reporter molecule such that a detectable positive signal is produced; and detecting the detectable positive signal, wherein detection of the detectable positive signal indicates a presence of one or more target molecules in the sample.
  • the methods further include distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system comprising a CRISPR-associated nucleas
  • Various embodiments of the invention provide a method of detecting a level and/or existence of a predefined nucleic acid sequence in a subject, which comprises (1) assaying a biological sample obtained from the subject, wherein the subject desires a determination regarding a disease, condition and/or pathogenic infection, said assaying comprising: preparing nucleic acid from the biological sample, amplifying the nucleic acid to generate copies of a template sequence, wherein the template sequence comprises a linear amplification promoter sequence and a target sequence, and contacting the copies of the template sequence to form a mixture with: a clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease, a guide RNA, said guide RNA recognizes the target sequence, a polymerase that recognizes the linear amplification promoter sequence, a plurality of ribonucleoside triphosphate (rNTP) if the template sequence is RNA-based, or a plurality of deoxyribose nucleo
  • Various embodiments provide a method of detecting a level and/or existence of a pathogen genomic material, comprising 1) assaying a biological sample obtained from a subject, who is optionally suspected of infection with the pathogen or desires a determination of an infection, and 2) detecting the level and/or existence of the detectable label as an indication of the predefined nucleic acid sequence.
  • the assaying is as described herein with a CRISPR-associated nuclease system.
  • the subject in the method desires a determination regarding an infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the predefined nucleic acid sequence comprises one or more portions of a gene encoding nucleocapsid protein (N gene), a gene encoding envelope (E gene), or both, of the SARS-CoV- 2
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the subject in the method desires a determined regarding a viral infection, such as one or more of infections caused by Coronavirus, SARS (including SARS-CoV-2 and its variants), Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zika virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Boma disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Human respiratory syncytial virus, Rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, Influenza, Hepatitis D virus, chickenpox virus, herpes virus, HIV, or hepatitis B virus; and the predefined nucleic acid sequence in the method (or the pathogen’s genomic material in the method) comprises at least a portion of
  • the preparation of nucleic acid comprises extracting RNA from the biological sample.
  • the amplification of the nucleic acid comprises reverse transcribing a portion of the RNA into the template sequence
  • said template sequence is DNA-based and comprises a T7 promoter sequence as the linear amplification promoter sequence
  • the polymerase that recognizes the linear amplification promoter sequence comprises T7 RNA polymerase.
  • the template sequence does not comprise a linear amplification promoter sequence; or the method of detection does not include a linear amplification step.
  • the CRISPR-associated nuclease comprises Casl3. In some embodiments of the method, the CRISPR-associated nuclease is Casl3a. In some embodiments of the method, the CRISPR-associated nuclease is Casl3b. In some embodiments the CRISPR-associated nuclease comprises Cas9, and the reporter molecule comprises a hairpin RNA structure and is annealed to the guide RNA; or wherein the CRISPR- associated nuclease comprises Casl2a, and the reporter molecule comprises a secondary DNA structure and is annealed to the guide RNA. In other embodiments, the CRISPR-associated nuclease is the nuclease in a CRISPR-Cas system described below.
  • the detectable label of the reporter molecule comprises a fluorescent dye and a quencher on opposing ends of the nucleotide sequence of the reporter molecule, wherein the quencher decreases fluorescence of the fluorescent dye when the nucleotide sequence is not cleaved, and the fluorescence increases when nucleotide sequence is cleaved, thereby separated from the quencher.
  • the reporter molecule operates via fluorescence, colorimetric dyes, or bioluminescence.
  • the reporter molecule is an oligonucleotide beacon described below.
  • the detection step comprises a visualization of the detectable label or of the reporter molecule, or a quantitative (or semi-quantitative) detection of the detectable label or of the reporter molecule.
  • the detection step includes visualizing the detectable label with a blue LED.
  • the detection step includes quantitatively measuring the detectable label signal in an equipment.
  • the detection step includes a lateral flow assay or test strip. Further embodiments of the detection step include one or more of those described below in Readout.
  • the method of detection is completed within 2 hours from preparation of the nucleic acid from the biological sample to the detection of the detectable label. In some embodiments, the method of detection is completed within 5 hours, 4 hours, 3 hours, 2 hours, 1 hour from preparation of the nucleic acid from the biological sample to the detection of the detectable label.
  • the method detects at least 10 copies of the template sequence per microliter of the mixture. In some embodiments, the method detects at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 copies of the template sequence per microliter of the mixture.
  • the preparation of the nucleic acid from the biological sample comprises extracting RNA from the biological sample in a lysis solution, wherein the lysis solution comprises octylphenoxypolyethoxyethanol (IGEPAL CA-630), sodium acetate, linear polyacrylamide in a HEPES-KOH buffer.
  • the lysis solution further comprises TCEP and glycerol. The components and content of the lysis solution is further described below.
  • Various embodiments provide a solution combination for extracting RNA and/or DNA from a biological sample, comprising a lysis solution for removing cell membranes and an isopropanol for precipitation.
  • a method of extracting RNA and/or DNA from a biological sample comprises incubating a mixture of a biological sample and a lysis solution, wherein the lysis solution comprises IGEPAL, HEPES, TCEP, glycerol, sodium acetate, and linear polyacrylamide, adding the mixture to isopropanol at a lowered temperature compared to temperature of the incubation step, thereby precipitating RNA and/or DNA of the biological sample, and removing the isopropanol to obtain precipitant, thereby extracting the RNA and/or the DNA from the biological sample.
  • the lysis solution comprises IGEPAL, HEPES, TCEP, glycerol, sodium acetate, and linear polyacrylamide
  • the mixture comprises the biological sample and the lysis solution at a volume ratio of 1 :2, and the biological sample comprises a nasopharyngeal swab, oropharyngeal swab, nasal swab, or buccal swab from a subject, and wherein the incubating is at ambient temperature or room temperature or about 20-35 °C for about 5 minutes, and the mixture is added to an ice-cold isopropanol.
  • the method further comprisese washing the precipitant with ethanol, drying the precipitant, and solubilizing the precipitant in nuclease-free water to obtain a solubilized precipitant; and optionally incubating the solubilized precipitant in a solution comprising DNase and proteinase to obtain the RNA, or incubating the solubilized precipitant in a solution comprising RNase and proteinase to obtain the DNA.
  • Exemplary CRISPR-Cas systems for use in the methods include one or more of Casl3 (including Casl3a, Casl3b, Casl3c), Cas9, and Casl2 (including subtypes Casl2a, Casl2b, Casl2c, Casl2g, Casl2h, Casl2i).
  • Exemplary Casl3a from different sources include L. buccalis (Lbu) Casl3a, Leptotrichia shahii (Lsh) Casl3a, Leptotrichiawadei (Lwa) Casl3a.
  • Casl3 is used in the methods or systems described in this invention. In some embodiments, Casl3a is used in the methods or systems described in this invention.
  • Casl3a a type VI- A CRISPR-Cas RNA-guided RNAribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA), and results in collateral RNA degradation. Specifically, the crRNA-target RNA duplex binds in a positively charged central channel of the nuclease lobe, and Casl3a protein and crRNA undergo a significant conformational change upon target RNA binding.
  • a “guide” refers to the guide region of crRNA, and this region is complementary, or substantially complementary (with exception being the first and/or last nucleotides), to target RNA.
  • the guide-target RNA duplex formation activates the HEPN catalytic site of Casl3a protein, (by triggering HEPN1 domain to move toward HEPN2 domain), which subsequently cleaves the bound target RNA beyond the guide complementary region and the free RNAs (“collateral”) in a non-specific manner. While Casl3a is activated by highly sequence-specific target binding, Casl3a can then cleave any RNA molecule in a non-specific manner. The nonspecifically cleaved “collateral” RNA, when modified with a detectable label, can serve as a reporter molecule, which reports the cleavage of the target RNA.
  • Casl3a, Cas9, Casl2a, and Casl2b are exemplary CRISPR-Case effectors for which ternary complex structures with guide RNA and target DNA (or RNA) are available. Yet, the catalytic site of Casl3a is located on the external surface, rather than internally as in Cas9 and Casl2. Casl3a can thus cleave any available ssRNA, functioning as a general non specific RNase, once it is activated by target RNA binding. In contrast, Cas9 and Casl2 are only able to cleave the target dsDNA complementary to the guide strand and in proximity to the HNH or RuvC catalytic sites.
  • Casl3a recognizes target RNA depending on the 3’-protospacer-flank site (PFS).
  • Casl3b is a type VI- B CRISPR-associated RNA-guided RNase, which is differentially regulated by accessory proteins like Csx27 and Csx28 (to avoid self-RNA cleavage).
  • a reporter molecule is an oligonucleotide beacon having at least one detectable label and an oligonucleotide sequence that is chosen according the cleavage preferences of the used Cas- protein.
  • the Cas-protein comprises LwaCasl3a
  • the reporter molecule is a ssRNA whose oligonucleotide sequence cleavable by the Cas-protein comprises poly-U.
  • the Cas-protein comprises LwaCasl3a
  • the oligonucleotide sequence cleavable by the Cas-protein comprises poly-U, e.g., 10 or more consecutive Uracil.
  • the oligonucleotide sequence cleavable by LwaCasl3a comprises 6 consecutive Uracil.
  • the oligonucleotide sequence cleavable by LwaCasl3a comprises 7 consecutive Uracil.
  • the oligonucleotide sequence cleavable by LwaCasl3a comprises 8 consecutive Uracil.
  • the oligonucleotide sequence cleavable by LwaCasl3a comprises 9 consecutive Uracil. In one embodiment, the oligonucleotide sequence cleavable by LwaCasl3a comprises 10-15 consecutive Uracil. In one embodiment, the oligonucleotide sequence cleavable by LwaCasl3a comprises 16-20 consecutive Uracil. In other embodiments, an oligonucleotide sequence cleavable by LbCasl2a is a ssDNA, comprising repeats of TTA, or repeats TTATT. By “repeats”, the number of repeat can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any integer.
  • Exemplary reporter types include but are not limited to fluorescence, colorimetric dyes, and bioluminescence.
  • the reporter molecule is fluorescence resonance energy transfer (FRET)-based, with a fluorophore and its quencher flanking on respective end of an oligonucleotide sequence that can be trans-cleaved (or collaterally cleaved) by an activated CRISPR-associated nuclease. Quenching of the fluorophore can occur as a result of the formation of a non-fluore scent complex between the fluorophore and another fluorophore or non-fluorescent molecule.
  • the reporter molecule is an oligonucleotide beacon, wherein the beacon has a fluorescent dye sitting on one end and a quencher on the other end.
  • the fluorescent dye When the molecule is cleaved, the fluorescent dye is separated from the quencher, thereby emitting fluorescent signal to be quantified with an instrument or a visualizer.
  • Fluorophores and their cognate quenchers are known in the art and can be selected for this purpose by one having ordinary skill in the art.
  • the reporter molecule is an oligonucleotide beacon, wherein the beacon has dual labels, one on each end.
  • a reporter molecule with 6- carboxy fluorescein (6-FAM) or FITC on the 5’ end and biotin on the 3’ end Recognition of target RNA or DNA sequence by the crRNA/Cas- or sgRNA/Cas- complex unleashes collateral activity, and the activated Cas protein cleaves the dual labeled reporter molecule, which leads to separation of Biotin- and FITC-/FAM-labels.
  • GNP gold nanoparticles
  • streptavidin is immobilized upstream on the test strip to indicate a control (C-) line position, which can capture the biotin fragment (when cleaved from the FITC- /F AM-labeled reporter fragment); and anti-goat antibody is immobilized downstream on the test strip to indicate a test (T-) line position, which can capture any GNPs with goat anti-FITC antibody that are not retained at the C-line.
  • the dual labeled reporter remains intact, and the biotin of the intact reporter molecule is bound at the C-line by streptavidin, so is the mobile anti-FITC conjugated GNPs by the FITC-/FAM-label in the reporter molecule.
  • the reporter molecule may one or more RNA oligonucleotides to which are attached one or more metal nanoparticles, such as gold nanoparticles, or quantum dots.
  • a plurality of metal nanoparticles is crosslinked by a plurality of RNA oligonucleotides forming a closed loop the cleavage of the RNA oligonucleotides by the CRISPR-associated nuclease leads to a detectable signal produced by the metal nanoparticles.
  • target sequences are exogenous, pathogen sequences - for detection or identification of an infection in a subject; endogenous, disease-related gene expression - for diagnosis of a disease or an infection in a subject, or a combination of the diseases or infections (multiplexing detection).
  • the methods are for use in detection of a nucleic acid sequence of SARS-CoV-2. In some embodiments, the methods are for use in detection of a nucleic acid sequence of SARS-CoV-2 in subject who desires a determination regarding COVID-19. In some embodiments, the methods are for use in detection of a nucleic acid sequence of SARS-CoV-2 in subject who exhibits symptoms of SARS-CoV-2 infection such as coughing, shortness of breath or difficult breathing, fever, chill, loss of smell or taste. In further embodiments, the methods are for use in diagnosing infection with SARS-CoV-2 in a subject.
  • Nucleic acid sequences of these pathogens are generally available in public database.
  • a predefined nucleic acid sequence to diagnose or identify SARS-CoV-2 can be the E (envelope) gene and/or the N (nucleoprotein) gene.
  • WWHO World Health Organization
  • US Centers for Disease Control and Prevention (CDC) provides information on assaying Nl, N2, and N3 region in the N gene.
  • the target sequence in the methods or the systems is the nucleocapsid protein (N) gene of SARS-CoV-2.
  • the target sequence in the methods or the systems is a portion (denoted Nl, N2, N3 etc.) of the nucleocapsid protein (N) gene of SARS-CoV-2.
  • the target sequence is an RNA sequence transcribed from template DNA, as shown in any one in Table 3, which includes a T7 RNA polymerase promoter and a sequence encoding one site of N gene of SARS-CoV-2.
  • N gene sequence can be obtained from the publicly available SARS-CoV-2 genome (GenBank accession no. MN908947). In some embodiments, a portion of the N gene refers to nucleotides 28303-28351, 29180-29228, or 28697-28748 of the genome location, as described in Lu et al., Emerg Infect Dis. , 2020;26(8): 1654-1665, which is incorporated by reference.
  • the Nl and N2 sites specifically detect SARS-CoV-2, and N3 universally detects all currently recognized clade 2 and 3 viruses within the subgenus Sarbecovirus, including SARS-CoV-2, SARS-CoV, and bat- and civet- SARS-like CoVs.
  • the target sequence in the methods or the systems is the Nl site in the SARS-CoV-2 nucleocapsid gene. In some embodiments, the target sequence in the methods or the systems is the N2 site in the SARS- CoV-2 nucleocapsid gene. In some embodiments, the target sequence in the methods or the systems is the N3 site in the SARS-CoV-2 nucleocapsid gene. In some embodiments, the target sequence in the methods or the systems includes both the Nl site and the N2 site in the SARS- CoV-2 nucleocapsid gene. In some embodiments, the target sequence in the methods or the systems includes both the Nl site and the N3 site in the SARS-CoV-2 nucleocapsid gene.
  • the target sequence in the methods or the systems includes both the N2 site and the N3 site in the SARS-CoV-2 nucleocapsid gene. In some embodiments, the target sequence in the methods or the systems includes the Nl, N2, and N3 sites in the SARS-CoV-2 nucleocapsid gene.
  • coronavirus sequences available from GenBank.
  • BLASTn analysis is performed (ncbi.nlm.nih.gov/blast/Blast.cgi) to ensure no major combined similarity of primers and probes with other pathogens that would potentially yield false-positive results (e.g., other coronaviruses (OC43, 229E, HKU1, NL63, and Middle East respiratory syndrome coronavirus [MERS-CoV]) or microflora of humans).
  • the target sequence (potential SARS-CoV-2 nucleotide sequence) is amplified with one or more techniques, thereby boosting the sensitivity in the detection methods. If starting with an RNA sample, reverse transcription can be performed to generate complementary DNA (cDNA) for amplification of specific DNA targets.
  • the methods include an amplification step by performing PCR, and preferably with Taq polymerase; rather than RPA. In other embodiments, recombinase polymerase amplification (RPA) is performed.
  • a suitable RNA or DNA amplification technique may include an isothermal amplification - such as nucleic-acid sequenced-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HD A), or nicking enzyme amplification reaction (NEAR), or a non-isothermal amplification - such as PCR, multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or ramification amplification method (RAM).
  • NASBA nucleic-acid sequenced-based amplification
  • RPA recombinase polymerase amplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HD A helicase-dependent amplification
  • NEAR nicking enzyme amplification reaction
  • a target DNA sequence is amplified at a constant temperature with a recombinase enzyme, a single-stranded DNA-binding protein (SSB), and a strand-displacing polymerase.
  • Recombinase enzymes first form a complex with a primer that scans the template DNA for complimentary sequences. Once found, the primer is annealed to the complimentary sequence and the non-complimentary template strand is displaced. The recombinase enzymes then disassociate from the primer and a DNA polymerase with a strand displacement activity, such as Bst DNA polymerase, binds to the double-stranded DNA formed by the primer and template.
  • a strand displacement activity such as Bst DNA polymerase
  • DNA single strand binding proteins attach to the displaced strand stabilizing the formation of a replication fork.
  • the DNA polymerase then extends the primer to produce a copy of the original template. Repeated extension of two opposing primers produces exponential amplification of the target DNA. While PCR relies on repeated heating and cooling cycles to denature and amplify DNA fragments, RPA is performed at a single moderate temperature (“isothermal”) and uses enzymatic activity to drive amplification. In various aspects, RPA boost the sensitivity of the detection, and it does not require expensive equipment, is less laborious, and therefore time-efficient. [0084] Various embodiments provide a linear amplification of the template sequence, which is achieved by introducing a promoter sequence into the template sequence.
  • RNA sample it can be initiated with reverse transcription of target RNA by a sequence-specific reverse primer to create a RNA/DNA duplex.
  • RNase H is then used to degrade the RNA template, allowing a forward primer containing a promoter, such as the T7 promoter, to bind and initiate elongation of the complementary strand, generating a double-stranded DNA product.
  • the RNA polymerase promoter-mediated transcription of the DNA template then creates copies of the target RNA sequence.
  • Each of the new target RNA sequence can be detected by the guide RNAs thus further enhancing the sensitivity of the assay.
  • T7 RNA polymerase can recognize the T7 promoter sequence when performing the transcription, and many copies of single-stranded RNA molecules can be produced.
  • the Casl3a-crRNA system includes a T7 polymerase, which transcribes target cDNA that also contains a T7 promoter sequence, thereby producing many copies of single-stranded target RNA, for cleavage (and therefore detection) by the Casl3a-crRNA system.
  • a T7 polymerase which transcribes target cDNA that also contains a T7 promoter sequence, thereby producing many copies of single-stranded target RNA, for cleavage (and therefore detection) by the Casl3a-crRNA system.
  • PCR polymerase chain reaction
  • the primers for PCR do not contain a T7 promoter, and the method does not include a linear amplification of the template sequence.
  • microfluidics-based devices/sy stems including microfluidic cartridges (‘chips’) that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection.
  • the systems further include, or can be used in combination with, a module for separating plasma from blood through a combined sedimentation-filtration effect.
  • the systems or devices are used in a multiplexing manner, i.e., parallel amplification and detection of multiple targets.
  • FIG. 1 For data analysis, control, and/or communication.
  • one or more instruments are used to detect the reporter labels from a sample.
  • reporter labels containing fluorescent molecules are detected using a qPCR instrument or a plate reader (e.g., Quantstudio5 qPCR).
  • reporter labels containing fluorescent molecules are detected visually via a P51TM molecular fluorescence viewer under the irradiation of a blue light.
  • reporter labels are detected with lateral flow test strips.
  • compositions for lysis of cells and/or extraction ofRNA, DNA, and/or protein from a biological sample comprises sodium acetate, polyacrylamide or acrylamide, octylphenoxypoly ethoxy ethanol (IGEPAL CA-630), and 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES).
  • IGEPAL CA-630 octylphenoxypoly ethoxy ethanol
  • HEPES 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid
  • a composition for lysis of cells and/or extraction ofRNA, DNA, and/or protein from a biological sample comprises sodium acetate, linear polyacrylamide (or acrylamide), IGEPAL CA-630, HEPES-KOH, and one or more of glycerol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and polyethylene glycol, in an aqueous medium of a pH ranging from 7.0 to 7.4.
  • a composition for lysis of cells and/or extraction of RNA, DNA, and/or protein from a biological sample comprises sodium acetate, linear polyacrylamide (or acrylamide), IGEPAL CA-630, HEPES-KOH, glycerol, and TCEP.
  • a composition for lysis of cells and/or extraction of RNA, DNA, and/or protein from a biological sample consists essentially of sodium acetate, linear polyacrylamide (or acrylamide), IGEPAL CA-630, HEPES-KOH, glycerol, and TCEP.
  • a composition for lysis of cells and/or extraction of RNA, DNA, and/or protein from a biological sample consists of sodium acetate, linear polyacrylamide (or acrylamide), IGEPAL CA-630, HEPES-KOH, glycerol, TCEP, and water.
  • a composition for lysis of cells and/or extraction of RNA, DNA, and/or protein from a biological sample comprises sodium acetate, linear polyacrylamide (or acrylamide), IGEPAL CA-630, HEPES- KOH, glycerol, and TCEP, and does not comprises DTT and PEG.
  • Various embodiments provide for a formulation for lysis of cells and/or extraction ofRNA, DNA, and/or protein from a biological sample, comprising sodium acetate at 300-600 mM, linear polyacrylamide at 30-80 pg/mL, IGEPAL CA-630 at 0.1%-1% (v/v), and HEPES potassium hydroxide (HEPES-KOH) in water with a pH from 7.0 to 7.4.
  • sodium acetate at 300-600 mM
  • linear polyacrylamide at 30-80 pg/mL
  • IGEPAL CA-630 at 0.1%-1% (v/v)
  • HEPES potassium hydroxide HEPES potassium hydroxide
  • the formulation further comprises glycerol at a concentration of glycerol at about 10% (v/v), TCEP at a concentration of about 20 mM, DTT at about 2 mM, polyethylene glycol 8000 at a concentration of about 10% (w/v).
  • composition or the formulation consists of, or consists essentially of, the sodium acetate, the polyacrylamide, the IGEPAL CA-630, the HEPES-KOH, water, and one or more of the glycerol, the TCEP, the DTT, and the polyethylene glycol.
  • a formulation of the lysis solution comprises IGEPAL CA-630 as the sole detergent to solubilize cell membrane (so does not include another detergent), TCEP as the sole reducing agent to destabilize nuclease (or DTT as the sole reducing agent; so does not include another reducing agent), HEPES or HEPES-KOH as the pH buffer to solubilize nucleic acid, sodium acetate and acrylamide (or linear polyacrylamide) to precipitate and as the co-precipitant, and glycerol as the crowding agent.
  • the sodium acetate in the composition or the formulation is 100- 150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, or 700-750 mM, or a combination thereof.
  • the sodium acetate in the composition or the formulation is 300-600 mM.
  • the sodium acetate in the composition or the formulation is 400-500 mM.
  • the sodium acetate in the composition or the formulation is 450 mM.
  • the polyacrylamide in the composition or the formulation is at a concentration of 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100 pg/mL, or a combination thereof. In one aspect, the polyacrylamide in the composition or the formulation is 30-80 pg/mL. In one aspect, the polyacrylamide in the composition or the formulation is 40- 60 pg/mL. In one aspect, the polyacrylamide in the composition or the formulation is 50 pg/mL.
  • acrylamide e.g., Bio-Rad #1610100
  • acrylamide is included in the composition or the formulation at 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100 pg/mL, preferably 30-80 pg/mL, more preferably 40-60 pg/mL
  • acrylamide is included in the composition or the formulation at 50 pg/mL.
  • the IGEPAL CA-630 in the composition or the formulation is at a concentration of 0.05-0.1%, 0.1-0.2%, 0.2-0.3%, 0.3-0.4%, 0.4-0.5%, 0.5-0.6%, 0.6-0.7%, 0.7- 0.8%, 0.8-0.9%, 0.9-1.0%, (v/v) or a combination thereof.
  • the IGEPAL CA-630 in the composition or the formulation is 0.3-0.8% (v/v).
  • the IGEPAL CA-630 in the composition or the formulation is 0.4-0.6% (v/v).
  • the IGEPAL CA-630 in the composition or the formulation is 0.5% (v/v).
  • the HEPES-KOH in the composition or the formulation is 2-5, 5- 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 mM, or a combination thereof. In one aspect, the HEPES-KOH in the composition or the formulation is 10-30 mM. In one aspect, the HEPES-KOH in the composition or the formulation is 20 mM. In one aspects, the HEPES-KOH in the composition or the formulation is at an effective amount for buffering the pH of the formulation or the composition to be about 7.2, or between 7.0 and 7.4.
  • the composition or formulation is used to lyse a cell from a sample, comprising incubating the sample with the composition or formulation in a volume ratio preferably of 1:2, or can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2.2, 1:2.4, 1:2.6, 1:2.8, or 1:3, for about 5 minutes at room temperature.
  • a volume ratio preferably of 1:2, or can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2.2, 1:2.4, 1:2.6, 1:2.8, or 1:3, for about 5 minutes at room temperature.
  • the incubation mixture is further precipitated on ice using cold isopropanol (or ethanol).
  • a further step can be included to incubate the precipitant (or washed precipitant) with a DNase to remove DNA.
  • a further step can be included to incubate the precipitant (or washed precipitant) with an RNase to remove RNA.
  • a handheld, hand-powered centrifuge which includes a rotor that has holes for insertion of microtubes (e.g., six holes for loading six 1.5ml microtubes), two or more driving strings that are coiled (e.g., supercoiled) and extend on two opposite ends beyond the rotor for pulling, and a locking-lid, wherein pointed or sharp edges are chamfered to prevent string abrasion, and the string holes are placed far away from the rotational axis to increase angular velocity.
  • the cover is placed on the centrifuge after tubes and collers have been inserted.
  • the correct lid orientation is 90-degree edge facing down, camphered edge facing up.
  • a dowel, locking pin, or bread tie can be used to secure the lid. Based on laser tachometer measurements this centrifuge achieves -3,500 RCF.
  • the average angular velocity can be increased by using a longer string. For example, 135 test braided fishing line of about one meter long can be used.
  • a system comprising a portable PCR machine, a portable fluorescence viewer, one or more individual discrete volumes, each individual discrete volumes comprising a CRISPR-associated nuclease, one or more guide RNAs designed to bind to corresponding target molecule, a reporter molecule, and optionally further comprising nucleic acid amplification reagents and a lysis solution.
  • This system can also be referred to as a CREST (Casl3-based, Rugged, Equitable, Scalable Testing) system.
  • MINIPCR® mini thermal cyclers are used in the amplification of nucleic acid from the biological sample to generate template sequences.
  • Mini thermal cyclers are portable, durable, bluetooth-enabled and compatible with Windows, Mac, Chromebooks, and Android; as well as compatible with standard PCR reagents and consumables.
  • an 8-well MINIPCR, mini8 thermal cycler is included in a system or a combination.
  • a 16-well MINIPCR, mini 16 thermal cycler is included in a system or a combination.
  • MINIPCR® mini thermal cyclers are used for a Taq polymerase-based amplification of nucleic acid in 10 cycles, 11 cycles, 12 cycles, 13 cycles, 14 cycles, 15 cycles, 16 cycles, 17 cycles, 18 cycles, 19 cycles, 20 cycles, 21 cycles, 22 cycles, 23 cycles, 24 cycles, or 25 cycles of thermal cycling.
  • MINIPCR® mini thermal cyclers are used for a Taq polymerase-based amplification of nucleic acid in no more than 20 cycles of thermal cycling.
  • thermocyclers can be used, including but are not limited to an Eppendorf thermocycler, a Biometra thermocycler, a Bio-Rad thermal cycler, a Perkin Elmer thermocycler, and an Applied Biosystems thermocycler.
  • a plastic-filter-based, LED visualizer is included in a system or a combination to aid visualization of a fluorescent signal.
  • a P51TM molecular fluorescence viewer is included in a system or a combination.
  • a quantitative fluorescence detection/measurement equipment is used, including but not limited to a fluorescence plate reader, a POCT analyzer quantitative fluorescence immunechromatography machine, and a quantitative fluorescence cytometry.
  • a system comprising one or more individual discrete volumes, each individual discrete volumes comprising a CRISPR- associated nuclease, one or more guide RNAs designed to bind to corresponding target molecule, a reporter molecule, and optionally further comprising nucleic acid amplification reagents.
  • a lysis solution is included in the system or the combination and is provided in one or more individual discrete volumes, each individual discrete volumes comprising sodium acetate, polyacrylamide, octylphenoxypolyethoxyethanol, and HEPES in water (e.g., nuclease-free water), and optionally further comprising glycerol, dithiothreitol, tris(2-carboxyethyl)phosphine, polyethylene glycol, or a combination thereof.
  • water e.g., nuclease-free water
  • the individual discrete volumes are droplets, or the individual discrete volumes are defined on a solid substrate, or the individual discrete volumes are microwells, or the individual discrete volumes are spots defined on a substrate, such as a paper substrate.
  • nucleic acid detection systems or methods described herein are designed to detect one or more viral targets and used in conjunction an anti-viral therapeutic and/or a standard-of-care treatment.
  • a treatment is administered to a subject detected with a presence (or existence) of SARS-CoV-2, and the treatment comprises steroids, corticosteroids, dexamethasone, vitamins (vitamin C, vitamin D), zinc, remdesivir, bamlanivimab, casirivimab, imdevimab, acetaminophen, or a combination thereof.
  • a method for treating SARS-CoV-2 infection in a subject comprising diagnosing SARS-CoV-2 infection by detecting a level or existence of SARS-CoV-2 virus as disclosed herein, and administering a treatment to the subject detected with the SARS-CoV-2 virus.
  • a method for treating SARS-CoV-2 infection in a subject comprising obtaining a result of an analysis of a level of SARS-CoV-2 virus as disclosed herein in a subject, and administering a treatment to the subject when the level of SARS-CoV-2 virus is higher than a negative control.
  • a method for treating SARS-CoV-2 infection in a subject comprising requesting a result of an analysis of a level of SARS-CoV-2 virus as disclosed herein in a subject, and administering a treatment to the subject when the level of SARS-CoV-2 virus is higher than a negative control.
  • a method for treating SARS-CoV-2 infection in a subject comprising administering a treatment to a subject who has been determined to have a level of SARS-CoV-2 virus higher than a negative control.
  • a negative control or a reference, can be a sample from a subject without SARS- CoV-2 infection, or a sample with heat-inactivated SARS-CoV-2 virus.
  • Various embodiments provide methods for diagnosing a disease, a condition, or a pathogenic infection in a subject, comprising detecting a level or existence of a predefined nucleic acid sequence as described herein, wherein the predefined nucleic acid sequence comprises genomic sequence or a portion thereof of a pathogen, or a gene marker associated with the disease or condition, and a level of the predefined nucleic acid sequence above a reference value indicates that the subject is diagnosed with the disease, the condition, or the pathogenic infection.
  • methods for diagnosis of SARS-CoV-2 infection in a subject include detecting a level or existence of N gene (Nl, N2, N3, or a combination thereof) or E gene of the SARS-CoV-2 from a mucus or saliva sample of a subject using a CRISPR-associated nuclease as described herein, and the detection of the N gene or the E gene above a reference value provides diagnosis of SARS-CoV-2 infection in the subject.
  • N gene N2, N3, or a combination thereof
  • E gene of the SARS-CoV-2 from a mucus or saliva sample of a subject using a CRISPR-associated nuclease as described herein
  • Example 1 A Scalable, Easy-to-Deploy, Protocol for Casl3-Based Detection of SARS- CoV-2 Genetic Material.
  • thermocyclers required for PCR are expensive, specialized instruments that are generally limited to a professional laboratory.
  • PCR is made accessible through the affordable, ultra-basic, Bluetooth-enabled, Peltier-device-based thermocyclers, which can even be battery operated. These versatile thermocyclers can be used in unconventional environments and perform as well as traditional thermocyclers in moderate temperatures.
  • RNA extraction kit QIAamp Mini Elute Vims Spin Kit, Qiagen. This RNA was used as input for a parallel comparison between CREST and the CDC- recommended one-step TaqMan assay. Considering that CREST was designed to provide a binary outcome, the CREST-to-TaqMan comparison was fitted to a sigmoid.
  • CREST was designed to be an accessible and scalable assay for detecting SARS-CoV-2, the study so far required RNA extraction using commercial kits.
  • PEARL Precipitation Enhanced Analyte RetrievaL
  • CREST was coupled to PEARL and found that commercial RNA extraction could be omitted (FIG. 6H).
  • Casl3a protein can be purified from a 1 -liter bacterial culture for use in more than 500,000 reactions, , which leads us to conclude its cost per reaction is minimal.
  • Casl3 protein can cost in the range of other enzymes used in this study, between $0.05 and $0.50 per reaction.
  • Other cost considerations include the labor involved in handling samples and setting up reactions.
  • Single strand DNA ultramers were purchased from Eton Biosciences (San Diego, CA, USA).
  • the ssDNA ultramers were dissolved in nuclease free water at a concentration of 10 pg/pL.
  • the ultramers were annealed in 2X annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) by heating at 95 °C for ten minutes in an aluminum heat block. While remaining in the heat block, the annealed ultramers were slowly cooled to room temperature. The annealed ultramers were subsequently separated on 4-20% gradient TBE PAGE gels (Thermo Fisher Scientific) at 150V.
  • Gels were stained with SYBR-SAFE (Thermo Fisher Scientific) diluted 1:10,000 in IX TBE buffer for 10 minutes, and the annealed DNA were excised from the gel using a clean razor blade while being visualized with a transilluminator.
  • Gel fragments were placed in 1.5 mL tubes with 500 pL TE buffer (10 mM Tris pH 8.5, 1 mM EDTA) and shaken overnight at 700 rpm, 16°C.
  • TE buffer containing the eluted DNA templates was removed from the gel fragment and placed into a fresh tube.
  • the annealed DNA templates were subsequently precipitated by mixing with one volume of ice-cold isopropanol and one ninth volume of 3 M sodium acetate pH 5.5, incubation at -80°C for thirty minutes, and collected via centrifugation at 20,000 x g (4°C) for thirty minutes. Pellets were washed with 1 mL ice-cold 80% ethanol and air-dried for ten minutes. The DNA was resuspended in 10 pL of nuclease free water. dsDNA quantity and purity were assessed using a NanoDrop ONE instrument (Thermo Fisher Scientific).
  • DNA templates were transcribed using the Megascript T7 Transcription kit (Thermo Fisher Scientific) 500 ng of PAGE-purified dsDNA were used as input for each reaction, all other reagents were added according to the manufacturer’s protocol. Transcription was allowed to proceed overnight at 37°C. After transcription, DNA templates were removed by incubating the reaction mixture with DNase at 37°C for 15 minutes. Ammonium acetate was added to inactivate the DNases according to the manufacturer’s protocol.
  • RNA targets were purified by phenol extraction. Briefly, the RNA was subjected to two sequential extractions, the first using one volume of acid phenol (5:1 phenol: chloroform, pH 4.5, Thermo Fisher Scientific) and a second one using one volume of chloroform. After addition of the organic solvents, the samples were mixed by vortex and centrifuged at 12,500 x g (4°C) for 15 minutes. The aqueous phase was recovered each time.
  • RNA-containing aqueous phase was mixed with one volume of ice-cold isopropanol and one ninth volume of 3M sodium acetate pH 5.5, incubated at -80°C for 20 minutes and the precipitated RNA was collected by centrifugation at 16,000 x g (4°C) for 20 minutes.
  • the RNA pellets were washed with 1 mL of ice-cold 80% ethanol and air dried for 10 minutes.
  • the RNA was resuspended in 10 pL of nuclease free water. RNA quantity and quality were assessed using aNanoDrop ONE instrument (Thermo Fisher Scientific).
  • Reverse transcription was performed using RevertAid Reverse Transcriptase (200 U/pL, Thermo Fisher Scientific) in presence of murine RNase inhibitor (New England Biolabs).
  • PCR polymerase chain reaction
  • RPA recombinase polymerase amplification
  • Target DNA molecules were amplified by PCR using a Taq DNA polymerase Master Mix (New England Biolabs).
  • thermocycler for amplification of target DNAs (can also be run in another thermocycler).
  • Target DNA molecules were amplified by RPA using TwistAmp Liquid Basic RPA kits (TwistDx, Maidenhead, UK).
  • Plasmids expressing Casl3a were used (pC013: Addgene #90097). Purification of Casl3a was performed according to published protocols (Kellner et al. Nature Protocol 2019). Briefly, Rosetta 2(DE3)pLysS cells (Millipore) harboring the Twinstrep-SUMO- huLwaCasl3a were grown in TB medium until an OD600 of 0.6 was achieved. Protein expression was induced by treating the cells with 250mM IPTG overnight at 16°C.
  • Casl3a was eluted from the resin, diluted to lower salt content, and loaded onto a cation exchange column equilibrated in 20 mM Tris pH 7.5, 24 mM NaCl, 5% Glycerol, 1 mM DTT. Fractions containing Casl3a were pooled, concentrated, and loaded onto an SEC200 column equilibrated in 50mM Tris pH 7.5, 600mMNaCl, 2mMDTT. Fractions containing Casl3a were pooled and buffer exchanged into Casl3a storage buffer (50 mM Tris pH 7.5, 600 mMNaCl, 5% Glycerol, 2 mM DTT). Protein concentration was determined by absorbance at 280 nm. Typical yields were 7 mg/L.
  • Casl3a was used for site-specific detection with three readouts; fluorescent detection using a Quantstudio5 qPCR instrument (Applied Biosystems), or using a P51 visualizer (miniPCR Bio), and visual detection with lateral flow strips. Fluorescent readouts followed similar protocols, and lateral flow strips utilized a different cleavage reporter that was 3’ biotinylated instead of the chemical addition of Iowa Black Quencher.
  • Casl3a detection was performed in Casl3a cleavage buffer (lx: 40 mM Tris pH 7.5, 1 mM DTT) supplemented with 1 mM rNTPs (Thermo Fisher Scientific), 2 U/pL RNase Inhibitor (New England Biolabs), 0.125 mM cleavage reporter (Integrated DNA Technologies), 1.5 U/pL T7 RNA Polymerase (Lucigen), 6.3 ng/pL LwaCasl3a, 20 nM Casl3 crRNA and 9 mM MgCb unless otherwise indicated.
  • TaqPath l-step RT-qPCR MasterMix (Cat #pdt A15300) was purchased from Applied Biosystems. A master mix of was prepared using the established CDC protocol. 15 pL of the master mix were dispensed into the qPCR plate before addition of input RNA. Serial dilutions of in vitro transcribed RNA, ranging from 200 fg/pL to 2 ag/pL and corresponding to 10 7 to 10 2 viral copies/ pL, were prepared in nuclease free water. 5 pL of each RNA target dilution was added to the wells containing the corresponding TaqMan primers and probes.
  • RNA extraction one can use CDC approved kits, or QIAamp minElute virus spin kit (cat# 57704) or QIAamp Viral RNA Mini Kit (cat# 52904); or can bypass RNA extraction as shown in Example 2);
  • Murine RNase inhibitor (40 U / pL) at 0.5 pL;
  • 10X Cleavage Buffer 400 mM Tris pH 7.5, 10 mM DTT) at 0.5 pL; rNTPs (25 mM each) at 0.2 pL;
  • Murine RNase Inhibitor (40 U/pL) at 0.25 pL;
  • Example 2 A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein to Facilitate the Detection of SARS-CoV-2.
  • Various embodiments of the invention take advantage of the distinct biochemical properties of biomolecules to selectively solubilize cellular components while partitioning nucleic acids (genetic material) into an insoluble fraction.
  • the method is a fast, easy, column- free, and cost-effective way to remove PCR inhibitors such as metal ions, salts, lipids, and antibiotics from samples, while simultaneously concentrating viral genetic material, enhancing the probability of detection in downstream analyses.
  • the lysis solution contains a non-ionic detergent such as octylphenoxypoly ethoxy ethanol (IGEPAL-CA-630) at a concentration of about 0.5% v/v to solubilize biological membranes and promote cell lysis.
  • the solution may also contain the crowding agent Propane- 1, 2,3 -triol (glycerol) at a concentration of about 20% v/v to enhance solubility of proteins and prevent their co-migration with nucleic acids during precipitation.
  • nucleic acids may be precipitated using 1 sample volume of 2-propanol.
  • all reagents are standard laboratory chemicals, and the only instruments required to make solutions are a scientific scale and pH meter.
  • the individual components of the solution may be prepared as high concentration stocks, then diluted to the working concentration in a single solution. Supporting data was developed and shown in figure 8.
  • Table 11 An exemplary formulation of PEARL lysis solution.
  • PEARL Precipitation Enhanced Analyte RetrievaL
  • PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins.
  • PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits.
  • PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.
  • RNA isolation a phenol- and guanidine-based reagent routinely used for isolation of RNA, DNA, and proteins.
  • TRIzol a phenol- and guanidine-based reagent routinely used for isolation of RNA, DNA, and proteins.
  • TRIzol extraction is labor intensive, which challenges scaling-up to meet testing demands, or in a recent study for a 5- minutes preparation, it requires expensive proprietary lysis solutions.
  • guanidium chloride was used for sample lysis in nasal swabs obtained from COVID- 19 positive patients, and total RNA was subsequently precipitated with isopropanol. Yet, the use of the toxic chaotropic agent guanidium chloride requires special disposal guidelines. Recently, direct detection of SARS-CoV-2 in nasopharyngeal swab samples without RNA extraction was reported, indicating that the initial RNA isolation step could be omitted. However, this approach results in reduced sensitivity of downstream quantitative PCR (qPCR)-based detection. On average, this approach required an additional 5-7 PCR cycles to reach the detection threshold when compared to reactions templated on purified RNA.
  • qPCR quantitative PCR
  • PEARL uses laboratory reagents (e.g., K-Hepes, NaOAc, Igepal CA-630, Glycerol, TCEP, polyacrylamide, isopropanol) to recover target analytes by precipitation. Briefly, a sample is mixed with PEARL lysis solution, which disrupts cell membranes and viral envelopes, liberating DNA, RNA, and proteins. These analytes are subsequently recovered by alcohol-based precipitation and centrifugation (FIG. 9A). The PEARL lysis solution contains the non-ionic detergent octylphenoxypoly ethoxy ethanol (IGEPAL-CA-630), which solubilizes biological membranes.
  • laboratory reagents e.g., K-Hepes, NaOAc, Igepal CA-630, Glycerol, TCEP, polyacrylamide, isopropanol
  • TCEP tris(2-carboxy ethyl) phosphine
  • Glycerol a low molecular weight crowding agent, together with sodium acetate and linear polyacrylamide (LPA) aid in precipitating DNA, RNA, and proteins.
  • RNA was used to examine the levels of the SARS-CoV-2 nucleoprotein (N) gene as well as the host RNaseP mRNA in the samples using the 1-step reverse transcription qPCR reference test for COVID-19 recommended by the United States Centers for Disease Control and Prevention (CDC) (TaqMan RNA-to-Ct 1-Step Kit, ThermoFisher).
  • CDC United States Centers for Disease Control and Prevention
  • Additive concentrations as follows: 10% glycerol, 2 mM DTT, 20 mM TCEP and 10% polyethylene glycol 8000 (PEG).
  • PEARL required a modest increase in initial sample input (1.25-fold) to achieve similar sensitivity to that of the commercial RNA extraction kit we used (Fig. 9B, note that the sample input for PEARL was either 175 pL or 250 pL, while the sample input for the commercial kit we used was 200 pL or 140 pL, respectively). Together, these results indicate that PEARL can be used as an alternative to commercial RNA extraction kits without substantial loss in sensitivity.
  • KSHV Zika virus
  • ZIKV Zika virus
  • PEARL extracts and probed for viral and host nucleic acids and proteins using qPCR- and immunodetection-based assays, respectively.
  • DNase I to detect RNA
  • RNase A to detect RNA
  • PEARL can facilitate the detection of mRNAs from DNA and RNA viruses.
  • the primers target sequences in different b-Actin exons, distinguishing mRNA amplicons from genomic DNA amplicons by molecular size.
  • PCR primers that amplify the non-transcribed promoter region of the host gene HSPA5.
  • PEARL Coupling PEARL to different downstream analyses for detection of nucleic acids and proteins can provide a powerful tool for detection of diverse viruses. Moreover, because RNA, DNA, and proteins are extracted at once, PEARL reduces sample handling time, allowing for streamlined diagnostic procedures. Thus, it may enable both nucleic acid and antigen-based SARS-CoV-2 testing. PEARL’s minimal handling requirements also make it scalable, which is desirable for high volume testing operations, as is needed for SARS-CoV-2 testing.
  • the collection medium used to store samples before processing may influence the performance of PEARL.
  • the viral transport media recommended by the CDC to store and inactivate samples for SARS-CoV-2 testing (2% Fetal Bovine Serum, 193 pg/mL Gentamicin, 0.5 pg/mL Amphotericin B, and various salts) has components that could co-precipitate with target analytes. Isopropanol is less polar than ethanol, and therefore, it has a higher propensity to precipitate salts and antibiotics. In our experiments, coprecipitation of salts and antibiotics does not appear to compromise downstream RT-qPCR or immunodetection assays.
  • PEARL uses common reagents and it does not require expensive equipment or highly trained personnel, it can provide an accessible alternative for streamlining diagnostics in geographic areas that lack access to capital, specialized reagents, and professional laboratories. Moreover, PEARL is field-deployable, given that a hand-powered centrifugation device can be used. In view of these considerations, coupling PEARL to our recently developed CRISPR-based protocol for detection of SARS-CoV-2 genetic material called CREST (Cas 13 -based, Rugged, Equitable, Scalable Testing) could allow efficient, affordable, widespread testing, lowering the barrier of “luxury testing” in many regions of the world.
  • CRISPR-based protocol for detection of SARS-CoV-2 genetic material called CREST (Cas 13 -based, Rugged, Equitable, Scalable Testing) could allow efficient, affordable, widespread testing, lowering the barrier of “luxury testing” in many regions of the world.
  • the precipitated material was collected by centrifugation at 19,000 x g for 10 minutes, washed once with 75% ethanol, air-dried for 5 minutes at room temperature and solubilized in 20 m ⁇ of nuclease-free water for amplification-based detection of nucleic acids or immunodetection of proteins.
  • RNA and protein were performed by nuclease/protease digestion and gel electrophoresis as follows: RNase A (Ambion), 5 pg/pl, 15 min at 37 °C; DNase I (New England Biolabs) 0.2 U/pl, 30 min at 37 °C; proteinase K (Macherey-Nagel), 2 pg/pl, 30 min at 37 °C.
  • RNase A Ambion
  • DNase I New England Biolabs
  • proteinase K Macherey-Nagel
  • Total RNA was isolated from PEARL extracts using TRIzol (Therm oFisher) following manufacturer’s recommendations.
  • iSLK-219 cells are latently infected with KSHV.219. This recombinant virus is maintained in cells as an episome. GFP is constitutively expressed from the episome, under the control of the human EF1 promoter. iSLK-219 cells also harbor the gene for a doxycycline-inducible KSHV RTA transcription activator.
  • Uninfected iSLK and KSHV-infected iSLK-219 cells were grown to 80% confluence, collected by trypsinization after two washes with PBS (GenClone), counted, and diluted at the desired density in 250 pL of PBS for PEARL extraction.
  • PBS GeneClone
  • ZIKV infections HeLa cells 245 were grown to 60% confluency and then infected with ZIKV at a multiplicity of infection (MO I) of 1. 48 hours post-infection, the cells were collected by trypsinization after two washes with PBS (GenClone), and counted. Cells were diluted in 250 pL of PBS for PEARL extraction.
  • PEARL extracts were obtained from de-identified human samples or cultured cells. SARS-CoV-2 positive human samples were heat-inactivated by incubation at 56 °C for 30 minutes before RNA extraction. RNA from these samples was obtained with the QIAamp Mini Elute Virus Spin Kit (Qiagen) following the manufacturer’s protocol, using 200 pL of sample input and eluting the purified RNA in 50 pL. PEARL extracts were prepared using 250 pL of SARS-CoV-2 positive human samples or a fixed number of cultured infected cells suspended in 250 pL of PBS.
  • PEARL extracts from cultured cells were treated with either DNase I (1 unit per every 8 pL of PEARL extract, New England BioLabs) or with RNase A (0.1 mg per every 8 pL of PEARL extract, Therm oFisher) in a final volume of 10 pL for 30 minutes at 37 °C. 5 pL of DNase-treated samples were reverse transcribed in a final volume of 10 pL using the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s protocol and diluted 5-fold in nuclease-free water before qPCR.
  • DNase I 1 unit per every 8 pL of PEARL extract, New England BioLabs
  • RNase A 0.1 mg per every 8 pL of PEARL extract, Therm oFisher
  • Target detection by qPCR was carried out with SYBR Select Master Mix (Applied Biosystems) using 2 pL of diluted cDNA as template, and following the manufacturer’s protocol.
  • SYBR Select Master Mix Applied Biosystems
  • the entire 10 pL from RNase-treated samples (genomic DNA) were diluted 5-fold with nuclease-free water and 2 pL of diluted genomic DNA were used as template for detection of specific genes with the SYBR Select Master Mix (Applied Biosystems) following the manufacturer’s protocol.
  • Detection of SARS- CoV-2 N 1 gene sequences and host RNase P mRNA from de-identified SARS-CoV-2 positive samples was carried out with the one-step TaqMan RNA-to-Ct 1-Step Kit (ThermoFisher), using 2 pL of undiluted PEARL extract, and following the manufacturer’s protocol. All qPCR data were collected using a CFX96 touch real time PCR instrument (Bio 270 Rad), and analyzed with the CFX Maestro 1.1 software (BioRad). Cq values were determined by regression. Data analysis and statistical tests were performed using the Graph Pad Prism 6.0 software.
  • Hand-powered centrifuge Our hand-powered centrifuge was designed in SolidWorks 2018 (Dassault Systemes), sliced (0.2 mm layer height) in Cura (Ultimaker), and printed on an Ender3 3D printer (Creality) using 1.75 mm polylactic acid filament (Hatchbox Inc). To actuate our device, we used Bru 294 tal Strong 135-test braided fishing line (Izorline International). Approximately 1 m of line was threaded through holes designed for the string- driven system in the hand pulls and, in the rotor, and the line was secured to itself with a double uni-knot forming a loop.
  • Izorline International Bru 294 tal Strong 135-test braided fishing line
  • RCF 1.118 x 10 5 x rmax x (RPM) 2 , where rmax is specified in centimeters.
  • 3D print files can be found at: 3dprint.nih.gov/discover/3dpx-014683. After PEARL precipitation, the samples were spun at maximum speed with the hand-powered centrifuge, or at 19,000 x g in a benchtop centrifuge. RNA and protein recovery for both centrifugation methods were determined by RT-qPCR and dot blot.
  • Example 3 Rapid CRISPR-based surveillance of SARS-CoV-2 in asymptomatic college students captures the leading edge of a community-wide outbreak.
  • Results All the 732 tests performed between late May to early June were negative. In contrast, tests performed on 1,076 samples collected between late June to early July revealed eight positive cases by CREST, confirmed by RT-qPCR and CLIA-diagnostic testing. The average age of the positive cases was 21.7 years; all individuals self-identified as students. These metrics showed that CREST was effective at capturing positive SARS-CoV-2 cases in our student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission.
  • UCSB UCSB’s population includes 26,134 students and 5,668 staff and faculty. 38% of the students live in university housing, and 34% in the nearby community of Isla Vista (23,096 residents, 1.866 mi 2 , 12,377 people/mi 2 ). This study was open to all symptom-free individuals, 18 years of age or older, affiliated with UCSB (student, faculty, staff, direct relatives). Individuals who exhibited a fever (100.4°F), cough, or shortness of breath in the two weeks before or on the day of sample collection were excluded from the study. Only five subj ects were excluded due to presenting symptoms at the time of collection and were referred to local healthcare resources.
  • UCSB healthcare professionals collected informed consent and demographic data (age, address, telephone, gender, and UCSB affiliation) at the sampling locale. Samples were assigned a numeric code for deidentification purposes. Samples were acquired as self- collected OP swabs stored in PBS, with surveillance by a healthcare professional. Samples were inactivated at 56 °C for 30 minutes, and RNA was extracted using the QIAamp MinElute Virus Spin Kit (Qiagen 57704) or Viral RNA Mini Kit (Qiagen 52906) from 140-200 pL of sample, and eluted in 50 pL. Pre- and post-analytical protocols were reviewed and approved by the Santa Barbara Cottage Hospital IRB.
  • Viral RNA was reverse transcribed and amplified using the TaqPath one-step cDNA master mix kit (ThermoFisher 501148245) following the manufacturer's recommendations. Briefly, a 15 pL master mix reaction was prepared using the established CDC protocol, and 5 pL of RNA were added into the reaction with each of the target- specific TaqMan primers and probes. For no template controls, 5 pL of nuclease-free water were used. Positive control reactions used 10 6 copies of in vitro transcribed RNA encoding the SARS- CoV-2 nucleocapsid sites N1 and N2.
  • RNAs are the same as in Example 1; and the CREST cleavage reporter herein is 6-FAM (Fluorescein) - (U)i4 (SEQ ID NO: 16) - BHQ (Blackhole quencher).
  • 6-FAM Fluorescein
  • U Ui4
  • BHQ Blackhole quencher
  • 5 pL of RNA were reverse transcribed using RevertAid (200 U/pL, ThermoFisher Scientific) in the presence of murine RNase inhibitor (NEB). Water was used as negative control. Positive control reactions used 10 6 copies of in vitro transcribed RNA.
  • the reaction mixtures were heated to 42 °C/30 minutes, then placed on ice.
  • 2 pL of the resulting cDNAs was used as templates for PCR amplification with Taq DNA polymerase (NEB), using the thermal profile: 98 °C/2 minutes; 20 cycles of 98 °C/15 seconds, 60 °C/15 seconds, and 72° C/15 seconds; final extension at 72°C/5 min.
  • Casl3a was used for site-specific detection using fluorescent probes.
  • the reaction was performed in Casl3a cleavage buffer (40 mM Tris pH 7.5, 1 mM DTT) supplemented with 1 mM rNTPs (ThermoFisher Scientific), 2 U /pL RNase Inhibitor (NEB), 0.125 mM cleavage reporter (IDT), 1.5 U/pL T7 RNA Polymerase (Lucigen), 6.3 ng/pL LwaCasl3a, 20 nM Casl3 crRNA and 9 mM MgCb.
  • the positive subjects average age was 21.7 years old, and all self-identified as UCSB students.
  • the eight samples detected by CREST were independently confirmed by a CLIA- certified laboratory test, and the results were reported to the participants and the Santa Barbara County Public Health Department (SBCPH) by Santa Barbara Cottage Hospital (SBCH) clinicians.
  • SBCPH Santa Barbara County Public Health Department
  • SBCH Santa Barbara Cottage Hospital
  • CRISPR-based strategy for large-scale viral surveillance in asymptomatic subjects.
  • This method uses PCR amplification and Casl3 for the detection of viral genomes with a simple binary outcome.
  • CREST is as efficient at detecting SARS-CoV-2 infections in asymptomatic subjects as the CDC recommended RT-qPCR, which is considered the “gold standard” testing method.
  • This CRISPR-based method also has the added benefit of enabling an easy to interpret and dependable binary readout, fluorescence vs. no fluorescence.
  • CREST showed perfect concordance with positive cases diagnosed in a CLIA certified laboratory (Pacific Diagnostics Laboratory), further corroborating its robustness. Because we designed CREST to be a low- cost and accessible method, it offers a much-sought alternative for communities where resources are limited, and where access to testing is difficult. Besides, CREST is scalable, enabling high throughput testing, and it uses laboratory-generated or off-the- shelf commercially available reagents, thus eliminating the restriction of limiting supply chains. For these reasons, we surmise CREST can offer a solution for places where access to professional laboratories is restrictive and instances in which a high volume of repetitive sampling is necessary, including the university setting.
  • Example 4 Comparison of SARS-CoV-2 screening using RT-qPCR or CRISPR-based assays in asymptomatic college students.
  • CRISPR-based methods offer reliable SARS-CoV-2 testing for virus screening and allow capture of the leading edge of an outbreak.
  • the mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR- based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures.

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Abstract

Le test évolutif d'une maladie ou d'une infection pathogène est limité par des obstacles de coût des réactifs, d'accessibilité aux instruments, de disponibilité de personnel hautement qualifié et d'un investissement initial élevé. L'invention concerne des systèmes et des procédés pour l'utilisation de tests robustes, équitables, évolutifs (CREST) à base de nucléase associé à CRISPR. CREST combine une réaction de polymérase en chaîne (PCR) avec une instrumentation à faible coût sans sacrifier la sensibilité de détection, et permet une interprétation binaire de résultats de détection. L'invention concerne en outre une nouvelle solution de lyse pour l'extraction d'acides nucléiques et/ou de protéine qui peut être utilisée pour préparer un échantillon d'acide nucléique dans un dosage CREST. Les systèmes et les réactifs de l'invention fournissent une solution de point d'intervention pour augmenter la distribution de la surveillance de la COVID-19.
PCT/US2021/028192 2020-04-20 2021-04-20 Système évolutif, facile à déployer et réactifs associés pour la détection à base de nucléase associée à crispr de matériel génétique pathogène WO2021216573A1 (fr)

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