Patent Application
20240002956
The present invention relates to methods for detection of pathogens. In particular, the disclosure relates to methods for detection of SARS-CoV-2 using a warm-start CRISPR assay.
Since its emergence in December 2019 (1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide, resulting in over one million deaths (2). As of now, fully validated vaccines and antiviral drugs are still unavailable. Therefore, methods for sensitive detection of this deadly virus and for quantification of viral loads in infected subjects are of upmost importance.
To detect and quantify SARS-CoV-2, TaqMan probe-based reverse transcription polymerase chain reaction (RT-PCR) is frequently used due to strong sensitivity and specificity (3, 4). However, this gold standard method greatly depends on expensive real-time quantitation PCR instruments and its quantitation accuracy is highly associated with well-designed TaqMan probes, not suitable for small clinics or community health settings. Alternatively, some isothermal nucleic acid amplification methods have been developed to rapidly detect SARS-CoV-2, such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) (5-7), reverse transcription recombinase polymerase amplification (RT-RPA) (8, 9), reverse transcription recombinase-aided amplification (RT-RAA) (10, 11), and sensitive splint-based one-pot isothermal RNA detection (SENSR) (12). However, these isothermal methods are inclined to qualitative detection or challenged by uncontrollable nonspecific amplification signals. Accordingly, what is needed are improved methods for accurate and sensitive detection of SARS-CoV-2 that are suitable for use in a variety of settings.
TO BE COMPLETED BY CASIMIR JONES UPON FINALIZATION OF THE CLAIMS
Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.
To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.
As used herein, the term “about,” when referring to a value or to an amount is meant to encompass variations of within +20% from that value or amount. In some embodiments, “about” refers to ±20%, +10%, ±5%, +1%, ±0.5%, or 0.1% from the specified amount, as such variations are appropriate.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of, cell and tissue culture, biochemistry, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
The term “amino acid” refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers, unless otherwise indicated, if their structures allow such stereoisomeric forms.
Natural amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), Lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V).
Unnatural amino acids include, but are not limited to, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, naphthylalanine (“naph”), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine (“tBuG”), 2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline (“hPro” or “homoP”), hydroxylysine, allo-hydroxylysine, 3-hydroxyproline (“3Hyp”), 4-hydroxyproline (“4Hyp”), isodesmosine, allo-isoleucine, N-methylalanine (“MeAla” or “Nime”), N-alkylglycine (“NAG”) including N-methylglycine, N-methylisoleucine, N-alkylpentylglycine (“NAPG”) including N-methylpentylglycine. N-methylvaline, naphthylalanine, norvaline (“Norval”), norleucine (“Norleu”), octylglycine (“OctG”), ornithine (“Orn”), pentylglycine (“pG” or “PGly”), pipecolic acid, thioproline (“ThioP” or “tPro”), homoLysine (“hLys”), and homoArginine (“hArg”).
As used herein, the term “artificial” refers to compositions and systems that are designed or prepared by man, and are not naturally occurring. For example, an artificial peptide or nucleic acid is one comprising a non-natural sequence (e.g., a nucleic acid or a peptide without 100% identity with a naturally-occurring protein or a fragment thereof).
As used herein, a “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another:
Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (or basic) (histidine (H), lysine (K), and arginine (R)); polar negative (or acidic) (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.
In some embodiments, unless otherwise specified, a conservative or semi-conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and/or amino acid analogs.
Non-conservative substitutions may involve the exchange of a member of one class for a member from another class.
The term “amino acid analog” refers to a natural or unnatural amino acid where one or more of the C-terminal carboxy group, the N-terminal amino group and side-chain functional group has been chemically blocked, reversibly or irreversibly, or otherwise modified to another functional group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.
The terms “complementary” and “complementarity” refer to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base-paring or other non-traditional types of pairing. The degree of complementarity between two nucleic acid sequences can be indicated by the percentage of nucleotides in a nucleic acid sequence which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementary). Two nucleic acid sequences are “perfectly complementary” if all the contiguous nucleotides of a nucleic acid sequence will hydrogen bond with the same number of contiguous nucleotides in a second nucleic acid sequence. Two nucleic acid sequences are “substantially complementary” if the degree of complementarity between the two nucleic acid sequences is at least 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100%) over a region of at least 8 nucleotides (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides), or if the two nucleic acid sequences hybridize under at least moderate, preferably high, stringency conditions. Exemplary moderate stringency conditions include overnight incubation at 37° C. in a solution comprising 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C., or substantially similar conditions, e.g., the moderately stringent conditions described in Sambrook et al., infra. High stringency conditions are conditions that use, for example (1) low ionic strength and high temperature for washing, such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50° C., (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin (BSA)/0.1% Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate at 42° C., or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at (i) 42° C. in 0.2×SSC, (ii) 55° C. in 50% formamide, and (iii) 55° C. in 0.1×SSC (preferably in combination with EDTA). Additional details and an explanation of stringency of hybridization reactions are provided in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York (1994).
The terms “crRNA” or “CRISPR RNA” are used interchangeably herein. The term crRNA is used in the broadest sense to cover any RNA involved in CRISPR methods, including pre-crRNA, tracrRNA, and guide RNA.
The “guide RNA,” “single guide RNA,” and “synthetic guide RNA,” are used interchangeably herein and refer to a nucleic acid comprising a crRNA containing a guide sequence. The terms “guide sequence,” “guide,” and “spacer,” are used interchangeably herein and refer to the about 20 nucleotide sequence within a guide RNA that specifies the target site. In CRISPR/Cas systems, the guide RNA contains an approximate 20-nucleotide guide sequence followed by a protospacer adjacent motif (PAM) that directs the endonuclease via Watson-Crick base pairing to a target sequence.
As used herein, a “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506, incorporated herein by reference), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000), incorporated herein by reference), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000), incorporated herein by reference), and/or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide” and “protein,” are used interchangeably herein.
As used herein, the term “percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Hence, in case a nucleic acid according to the technology is longer than a reference sequence, additional nucleotides in the nucleic acid, that do not align with the reference sequence, are not taken into account for determining sequence identity. Methods and computer programs for alignment are well known in the art, including BLAST, Align 2, and FASTA.
The term “sample” is used herein in the broadest sense and refers to any suitable sample, including liquids, solids, and gases. In some embodiments, the sample is a biological sample (e.g. a sample obtained from a subject). The biological sample may comprise a fluid sample or a tissue sample. In some embodiments, the biological sample is a blood sample or a blood product such as serum or plasma. In some embodiments, the sample comprises urine. In embodiments, the sample is a respiratory specimen, including a nasal sample (e.g. a nasal swab), a nasopharyngeal sample (e.g. a nasopharyngeal swab), an oropharyngeal sample (e.g. an oropharyngeal swab), a mid-turbinate sample (e.g. a mid-turbinate swab), sputum, endotracheal aspirate or bronchoalveolar lavage. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, the sample is a saliva sample. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is obtained from a subject suspected of having a viral infection. In some embodiments, the sample is obtained from a subject suspected of having an upper respiratory infection. In some embodiments, the sample is obtained from a subject suspected of having a SARS-CoV-2 infection. In some embodiments, the subject is a human. The sample can be used directly as obtained from a patient or can be pre-treated, such as by heating, filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like.
“Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In some embodiments, the subject is a human.
The terms “target sequence,” “target nucleic acid,” and “target site” are used interchangeably herein to refer to a polynucleotide (nucleic acid, gene, chromosome, genome, etc.) to which a guide sequence (e.g., a guide RNA) is designed to have complementarity, wherein hybridization between the target sequence and a guide sequence promotes the formation of a Cas/CRISPR complex, provided sufficient conditions for binding exist. In some embodiments, the target sequence is a viral nucleic acid sequence. In some embodiments, the target sequence is a SARS-CoV-2 sequence.
In some aspects, provided herein are primers. In some embodiments, provided herein are primers for amplifying SARS-CoV-2 nucleic acid. In some embodiments, provided herein is a composition comprising a plurality of primers for amplifying SARS-CoV-2 nucleic acid.
In some embodiments, provided herein is a primer having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 1. In some embodiments, provided herein is a primer having an amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, provided herein is a primer having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 2. In some embodiments, provided herein is a primer having an amino acid sequence set forth in SEQ ID NO: 2.
In some embodiments, provided herein is a primer having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 3. In some embodiments, provided herein is a primer having an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, provided herein is a primer having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 5. In some embodiments, provided herein is a primer having an amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, provided herein is a phosphorothioated primer. In some embodiments, provided herein is a primer having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a primer having an amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, provided herein is a primer having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, provided herein is a primer having an amino acid sequence set forth in SEQ ID NO: 6.
In some embodiments, provided herein is a primer having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, provided herein is a primer having an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, provided herein is a primer having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, provided herein is a primer having an amino acid sequence set forth in SEQ ID NO: 8.
In some embodiments, provided herein is a composition comprising a primer having at least 90% sequence identity to SEQ ID NO: 1, a primer having at least 90% sequence identity to SEQ ID NO: 2, a primer having at least 90% sequence identity to SEQ ID NO: 4, a primer having at least 90% sequence identity to SEQ ID NO: 6, a primer having at least 90% sequence identity to SEQ ID NO: 7, and a primer having at least 90% sequence identity to SEQ ID NO: 8.
In some embodiments, provided herein is a composition comprising a primer having the amino acid sequence set forth in SEQ ID NO: 1, a primer having the amino acid sequence set forth in SEQ ID NO: 2, a primer having the amino acid sequence set forth in SEQ ID NO: 4, a primer having the amino acid sequence set forth in SEQ ID NO: 6, a primer having the amino acid sequence set forth in SEQ ID NO: 7, and a primer having the amino acid sequence set forth in SEQ ID NO: 8. The composition may further comprise additional reagents, including those described in the warm-start CRISPR reaction mixture of section 3. Suitable additional reagents include, for example, a Cas endonuclease, a target-specific crRNA, pyrophosphatase, a reverse transcriptase, a DNA polymerase, and the like, including the specific reagents and amounts thereof described below.
In some aspects, provided herein are assays. In some embodiments, provided herein is a digital warm-start assay for detecting a target in a sample. In some embodiments, the warm-start digital assay for detecting a target in a sample comprises contacting a sample with a warm-start CRISPR reaction mixture, partitioning the warm-start CRISPR reaction mixture into a plurality of microwells, amplifying the target, if present in the sample, and detecting a signal in each of the plurality of microwells. Detection of the signal in a given microwell indicates the presence of the target in the microwell.
In some embodiments, the warm-start digital assay for detecting a target in a sample comprises contacting providing a warm-start CRISPR reaction mixture and partitioning the warm-start CRISPR reaction mixture into a plurality of microwells, wherein each microwell comprises a sample. In some embodiments, the assay further comprises amplifying the target, if present in the sample, and detecting a signal in each of the plurality of microwells. Detection of the signal in a given microwell indicates the presence of the target in the microwell.
In some embodiments, the target is amplified by isothermal amplification. In some embodiments, the assays described herein take advantage of isothermal amplification techniques to amplify the target, if present in the sample, and CRISPR/Cas technology to detect the target, if present. Accordingly, in some embodiments the warm-start CRISPR reaction mixture comprises a Cas endonuclease and a target-specific crRNA. The Cas endonuclease may be Cas9, Cas12a (Cpf1), or Cas13. In some embodiments, the endonuclease is Cas12a. The target-specific crRNA depends on the intended target to be detected in the sample. Generally speaking, the Cas endonuclease (e.g. Cas12a) and the target-specific crRNA form a complex. For example, as shown in
In some embodiments, the target is a nucleic acid. In some embodiments, the target is nucleic acid from a pathogen. For example, in some embodiments the target is viral nucleic acid. For example, the target may be viral RNA. In some embodiments, the target comprises viral nucleic acid from a virus causing upper respiratory infection. In some embodiments, the target comprises viral nucleic acid from an upper respiratory pathogen selected from SARS-CoV2, coronavirus, rhinovirus, influenza, respiratory syncytial virus, adenovirus, parainfluenza, human immunodeficiency virus, human papillomavirus, rotavirus, hepatitis C virus, zika virus, Ebola virus, tuberculosis, Borrelia burgdorferi, Staphylococcus, aspergillus, Streptococcus. pyogenes.
In some embodiments, the target is SARS-CoV-2 nucleic acid. In some embodiments, the target-specific crRNA comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9. For example, in some embodiments the target-specific crRNA comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the target-specific crRNA comprises the nucleotide sequence of SEQ ID NO: 9.
In some embodiments, the warm-start CRISPR reaction mixture further comprises primers for amplification of the target. In some embodiments, the warm-start CRISPR reaction mixture comprises primers for isothermal amplification of the target. In some embodiments, the warm-start CRISPR reaction mixture comprises primers for dual-priming isothermal amplification of the target (DAMP). DAMP is described in Ding et al., Anal. Chem. (2019) 91:20; 12852-12858, the entire contents of which are incorporated herein by reference for all purposes. In some embodiments, the warm-start CRISPR reaction mixture comprises outer primers and inner primers. In some embodiments, the warm-start CRISPR reaction mixture comprises a forward outer primer and a reverse outer primer. In some embodiments, the outer primers are 10-40 contiguous nucleotides in length. For example, the forward outer primer and/or the reverse outer primer may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides in length. In some embodiments, the warm-start CRISPR reaction mixture further comprises a forward inner primer and reverser inner primer. In some embodiments, the inner primers are 20-60 contiguous nucleotides in length. For example, in some embodiments the forward inner primer and/or the reverse inner primer are 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 contiguous nucleotides in length.
The primers are designed to amplify the target (e.g. the target nucleic acid sequence). Accordingly, appropriate selection of primers depends on the target itself. In some embodiments, the target is SARS-CoV-2. In some embodiments, the target is or is present within the nucleoprotein (N) gene of SARS-CoV-2. Accordingly, the primers may be designed to amplify the desired portion of the N gene of SARS-CoV-2. Other suitable primers may be designed for amplification of other desired targets. For example, the target may be viral nucleic acid, and the primers designed for amplification of said viral nucleic acid. For example, primers may be designed and used for amplification of viral nucleic acid selected from SARS-CoV2, coronavirus, rhinovirus, influenza, respiratory syncytial virus, adenovirus, parainfluenza, human immunodeficiency virus, human papillomavirus, rotavirus, hepatitis C virus, zika virus, Ebola virus, tuberculosis, Borrelia burgdorferi, Staphylococcus, Aspergillus, and Streptococcus Pyogenes nucleic acid.
In some embodiments, the warm-start CRISPR reaction mixture comprises a forward outer primer and a reverse outer primer, wherein the forward outer primer comprises an nucleotide sequence having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 1 and wherein the reverse outer primer comprises an nucleotide sequence having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 2. In some embodiments, the forward outer primer comprises an nucleotide sequence set forth in SEQ ID NO: 1 and the reverse outer primer comprises an amino acids sequence set forth in SEQ ID NO: 2.
In some embodiments, the warm-start CRISPR reaction mixture comprises inner primers. In some embodiments, the warm-start CRISPR reaction mixture comprises a forward inner primer comprising a nucleotide sequence having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 3 and an outer primer comprising a nucleotide sequence having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 5.
In some embodiments, the forward inner primer and/or the reverse inner primer is phosphorothioated. In some embodiments, the forward inner primer and the reverse inner primer is phosphorothioated. A “phosphorothioated primer” or a primer that “is phosphorothioated” refers to a primer in which at least one nucleotide comprises a phosphorothioate modification. For example, a phosphorothioated primer may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 nucleotides that are a phosphorothioated. In some embodiments, the nucleotides that are phosphorothioated are contiguous. For example, the primer may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 contiguous nucleotides that are a phosphorothioated. In some embodiments, a phosphorothioated primer comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 27, 29, or 30 nucleotides that are phosphorothioated. In some embodiments, the inner primers comprise a forward inner primer comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4, and the reverse inner primer comprises an nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the forward inner primer comprises a nucleotide sequence set forth in SEQ ID NO: 4, and the reverse inner primer comprises an nucleotide sequence set forth in SEQ ID NO: 6.
In some embodiments, the warm-start CRISPR reaction mixture further comprises a forward competition primer and a reverse competition primer. The competition primers may be added to mediate pair-priming strand extension. In some embodiments, the competition primers overlap with the inner primers, but are shorter than the inner primers. For example, in some embodiments, the forward competition primer comprises at least 10 contiguous nucleotides also present within the sequence of the forward inner primer, wherein the forward competition primer is shorter than the forward inner primer. For example, in some embodiments the forward competition primer comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides also present within the sequence of the forward inner primer. In some embodiments, the forward competition primer comprises 15-25 contiguous nucleotides present within the nucleotide sequence of the forward inner primer. In some embodiments, the forward competition primer comprises a nucleotide sequence having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 7. In some embodiments, the forward competition primer comprises the nucleotide sequence set forth in SEQ ID NO: 7.
In some embodiments, the reverse competition primer comprises at least 10 contiguous nucleotides also present within the sequence of the reverse inner primer, wherein the reverse competition primer is shorter than the reverse inner primer. For example, in some embodiments the reverse competition primer comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides also present within the sequence of the reverse inner primer. In some embodiments, the reverse competition primer comprises 15-25 contiguous nucleotides present within the nucleotide sequence of the reverse inner primer. In some embodiments, the reverse competition primer comprises a nucleotide sequence having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 8. In some embodiments, the reverse competition primer comprises the nucleotide sequence set forth in SEQ ID NO: 8.
In some embodiments, the warm-start CRISPR reaction mixture further comprises pyrophosphatase. In some embodiments, the warm-start CRISPR reaction mixture comprises less than 0.4 U/μl pyrophosphatase (concentration relative to the total volume of the warm-start CRISPR reaction mixture, not including the sample). For example, in some embodiments the warm-start CRISPR reaction mixture comprises about 0.01 U/μl to about 0.39 U/μl pyrophosphatase. In some embodiments, the warm-start CRISPR reaction mixture comprises less than about 0.39 U/μl, less than about 0.38 U/μl, less than about 0.37 U/μl, less than about 0.36 U/μl, less than about 0.35 U/μl, less than about 0.34 U/μl, less than about 0.33 U/μl, less than about 0.32 U/μl, less than about 0.31 U/μl, or less than about 0.3 U/μl pyrophosphatase. In some embodiments, the warm-start CRISPR reaction mixture comprises about 0.29 U/μl, about 0.28 U/μl, about 0.27 U/μl, about 0.26 U/μl, about 0.25 U/μl, about 0.24 U/μl, about 0.23 U/μl, about 0.22 U/μl, about 0.21 U/μl, about 0.2 U/μl pyrophosphatase. In some embodiments, the warm-start CRISPR reaction mixture comprises about 0.2 U/μl pyrophosphatase.
In some embodiments, the warm-start CRISPR reaction mixture further comprises a DNA polymerase. In some embodiments, the DNA polymerase comprises Bst DNA polymerase. In some embodiments, the warm-start CRISPR reaction mixture comprises less than about 36 U/μl (concentration relative to the total volume of the warm-start CRISPR reaction mixture, not including the sample) of the DNA polymerase (e.g. Bst DNA polymerase). In some embodiments, the warm-start CRISPR reaction mixture comprises about 1 U/μl to about 35 U/μl of the DNA polymerase (e.g. Bst DNA polymerase). In some embodiments, the warm-start CRISPR reaction mixture comprises about 1 U/μl, about 2 U/μl, about 3 U/μl, about 4 U/μl, about 5 U/μl, about 6 U/μl, about 7 U/μl, about 8 U/μl, about 9 U/μl, about 10 U/μl, about 11 U/μl, about 12 U/μl, about 13 U/μl, about 14 U/μl, about 15 U/μl, about 16 U/μl, about 17 U/μl, about 18 U/μl, about 19 U/μl, about 20 U/μl, about 21 U/μl, about 22 U/μl, about 23 U/μl, about 24 U/μl, about 25 U/μl, about 26 U/μl, about 27 U/μl, about 28 U/μl, about 29 U/μl, about 30 U/μl, about 31 U/μl, about 32 U/μl, about 33 U/μl, about 34 U/μl, or about 35 U/μl DNA polymerase (e.g. Bst DNA polymerase).
In some embodiments, the warm-start CRISPR reaction mixture further comprises a reverse transcriptase. In some embodiments, the warm-start CRISPR reaction mixture further comprises at least 0.1 U/μl of the reverse transcriptase. In some embodiments, the warm-start CRISPR reaction mixture further comprises at least about 0.1 U/μl, at least about 0.2 U/μl, at least about 0.5 U/μl, at least about 1 U/μl, at least about 1.5 U/μl, at least about 2 U/μl, at least about 2.5 U/μl, or at least about 3 U/μl reverse transcriptase.
In some embodiments the CRISPR-reaction mixture additionally comprises a reporter molecule. In some embodiments, the reporter molecule generates a signal, wherein the signal is detected to determine the presence and/or amount of target within the sample. For example, in some embodiments the reporter molecule comprises a fluorescent moiety conjugated to a quencher. In an intact (e.g. uncleaved) state, the fluorescence from the fluorescent moiety is quenched, such as by resonance energy transfer. However, after cleavage of the reporter molecule, the fluorescent moiety is released from the quencher and the fluorescent signal becomes detectable. In some embodiments, the reporter molecule comprises a single stranded DNA reporter. In some embodiments, the reporter molecule comprises a single-stranded, non-target DNA sequence labeled with a fluorescent label. For example, the reporter molecule may comprise a single-stranded, non-target DNA sequence labeled with fluorescein or derivatives thereof (e.g., 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)). The label may be present at the 5′ end or the 3′ end of the non-target DNA sequence. In some embodiments, the non-target DNA sequence comprises 2-10 amino acids. For example, the non-target DNA sequence may comprise 2-10 cysteine residues. For example, the non-target DNA sequence may comprise five cysteine residues (CCCCC; SEQ ID NO: 18).
In some embodiments, the endonuclease indiscriminately cleaves reporter molecules during cleavage of the amplicon generated as a result of isothermal amplification. Accordingly, binding of the Cas-crRNA complex (e.g. cas12a-crRNA complex) to the target amplicon induces cleavage of the reporter molecule. In contrast, in the absence of the target amplicon the endonuclease (e.g. cas12a) does not cause substantial (e.g. detectable) cleavage of the reporter molecule.
The warm-start CRISPR reaction mixture may further comprise additional reagents. For example, the warm-start CRISPR reaction mixture may comprise additional reagents to stabilize the sample (e.g. preservatives, inhibitors, etc.), along with suitable buffers, salts, dNTPs, and the like required for isothermal amplification and detection of the target, if present in the sample.
In some embodiments, the method comprises partitioning the warm-start CRISPR reaction mixture into a plurality of microwells. In some embodiments, the warm-start CRISPR reaction mixture has been contacted with the sample prior to partitioning. In other embodiments, the sample is present within the microwells. The method further comprises amplifying the target, if present in the sample, by isothermal amplification. In some embodiments, isothermal amplification comprises incubating the warm-start CRISPR reaction mixture at a temperature of about 50° C.-60° C. for at least about 10 minutes. For example, isothermal amplification may comprise incubating at a temperature of about 50° C.-60° C. for at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, or at least about 90 minutes. In some embodiments, isothermal amplification comprises incubating at a temperature of about 50° C.-60° C. for about 90 minutes. In some embodiments, the temperature is about 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., or 60° C. In some embodiments, the temperature is about 52° C.
In some embodiments, the method comprises signal in each of the plurality of microwells. For example, as described above incubation of the warm-start CRISPR reaction mixture following contact with the sample results in amplification of the target, if present in the sample. Using the primers described herein, an amplicon is generated that is bound by the endonuclease-crRNA complex (e.g. the Cas12a-crRNA complex). Binding of the complex allows for the endonuclease (e.g. cas12a) to also indiscriminately cleave the reporter molecule, thus generating a detectable signal (e.g. a detectable fluorescent signal). Detection of the signal in a well indicates the presence of the target in the well. In some embodiments, the number of detectable microwells can be quantified and used to determine the amount (e.g. level) of target in the sample.
The sample may be any suitable sample. In some embodiments, the sample is a biological sample (e.g. a sample obtained from a subject). The biological sample may comprise a fluid sample or a tissue sample. In some embodiments, the biological sample is a blood sample or a blood product such as serum or plasma. In some embodiments, the sample comprises urine. In embodiments, the sample is a respiratory specimen, including a nasal sample, an oropharyngeal sample, a mid-turbinate sample, sputum, endotracheal aspirate or bronchoalveolar lavage. In some embodiments, the sample is a cerebrospinal fluid sample. In some embodiments, the sample is a saliva sample. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is obtained from a subject suspected of having a SARS-CoV-2 infection. In some embodiments, the subject is a human. The sample can be used directly as obtained from a patient or can be pre-treated, such as by heating, filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like.
The disclosure further provides kits. In some embodiments, provided herein is a kit comprising one or more primers, compositions, or warm-start CRISPR reaction mixtures described herein. In some embodiments, provided herein is a kit comprising a primer having at least 90% sequence identity to SEQ ID NO: 1, a primer having at least 90% sequence identity to SEQ ID NO: 2, a primer having at least 90% sequence identity to SEQ ID NO: 4, a primer having at least 90% sequence identity to SEQ ID NO: 6, a primer having at least 90% sequence identity to SEQ ID NO: 7, and a primer having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the kit comprises a primer having the amino acid sequence set forth in SEQ ID NO: 1, a primer having the amino acid sequence set forth in SEQ ID NO: 2, a primer having the amino acid sequence set forth in SEQ ID NO: 4, a primer having the amino acid sequence set forth in SEQ ID NO: 6, a primer having the amino acid sequence set forth in SEQ ID NO: 7, and a primer having the amino acid sequence set forth in SEQ ID NO: 8.
In some embodiments, the kit further comprises additional reagents useful for isothermal amplification of nucleic acid. For example, the kit may further comprise a DNA polymerase, a reverse transcriptase, pyrophosphatase, dNTPs, buffers, salts, stabilizers, etc.
In some embodiments, the kit further comprises reagents for CRISPR-based detection of the isothermally amplified nucleic acid. For example, the kit may further comprise a reporter molecule. In some embodiments, the reporter molecule comprises a single-stranded, non-target DNA sequence labeled with a fluorescent label. For example, the reporter molecule may comprise a single-stranded, non-target DNA sequence labeled with fluorescein or derivatives thereof (e.g., 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)). The label may be present at the 5′ end or the 3′ end of the non-target DNA sequence. In some embodiments, the non-target DNA sequence comprises 2-10 amino acids. For example, the non-target DNA sequence may comprise 2-10 cysteine residues. For example, the non-target DNA sequence may comprise five cysteine residues (CCCCC (SEQ ID NO: 18)). In some embodiments, the kit further comprises an endonuclease (e.g. Cas12a). In some embodiments, the kit further comprises a target-specific cr-RNA. In some embodiments, the target-specific crRNA comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9. For example, in some embodiments the target-specific crRNA comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the target-specific crRNA comprises the nucleotide sequence of SEQ ID NO: 9.
The kit may be used for methods of detecting a target in a sample. In some embodiments, the target is nucleic acid. In some embodiments, the target is viral nucleic acid. For example, the target may be nucleic acid from an upper respiratory pathogen. For example, the kit may be used in methods of detecting SARS-CoV-2 nucleic acid in a sample. The kit may comprise additional components, including tubes (e.g. sample collection tubes, storage tubes, reaction tubes), buffers, stabilizers, salts, controls, calibrators, and the like. The kit may additionally comprise instructions for use. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Presented herein is a digital warm-start CRISPR (WS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical COVID-19 samples. The digital WS-CRISPR assay was established through partitioning a one-pot WS-CRISPR reaction into sub-nanoliter aliquots using commercial QuantStudio 3D digital chips. This reaction combined the reverse transcription dual-priming isothermal amplification (RT-DAMP) (23) and CRISPR-Cas12a-based fluorescence detection in one-pot format and is efficiently initiated at 52° C., the firstly reported digital warm-start CRISPR-Cas12a assay. Thus, the digital WS-CRISPR thoroughly addresses premature target amplifications at room temperature. Through targeting the SARS-CoV-2's nucleoprotein (N) gene, DWS-CRISPR is able to detect down to 50 copies/μl SARS-CoV-2 RNA in the sample, showing high sensitivity. In addition, digital WS-CRISPR is validated by quantitatively determining 32 clinical swab samples and 3 clinical saliva samples. More importantly, digital WS-CRISPR can directly detect SARS-CoV-2 in heated saliva samples without RNA extraction. This digital WS-CRISPR method is therefore a reliable, sensitive and straightforward CRISPR assay which facilitates SARS-CoV-2 detection toward digitized quantification.
As shown in
The WS-CRISPR assay combines RT-DAMP amplification with CRISPR-Cas12a detection in one-pot. The DAMP amplification is one variant of loop-mediated isothermal amplification using a new primer design strategy (23). Each inner primer of the DAMP amplification is formed with two target sites with a distance of below 40 nt and a pair of competition primers is added to mediate pair-priming strand extension (
There remains a challenge to directly couple LAMP or DAMP amplification with CRISPR-Cas12a detection in a “one-pot” format due to the significant difference in their reaction buffer compositions and reaction temperatures. For example, one of the major concerns is the concentration of Mg2+. The cleavage of Cas12a nucleases for both on-target and collateral activity is typically high-Mg2+-dependent (25-27), while not for LAMP or DAMP amplification. To enable high sensitive nucleic acid detection, different Cas12a nucleases were evaluated and compared evaluated at various concentration of Mg2+. As shown
Most Cas12a nucleases have an optimal activity at 37° C., but LAMP/DAMP amplification powered by Bst DNA polymerase typically requires high temperature of 60-65° C. (23, 28). To develop a “one-pot” assay, phosphorothioated inner primers of FI an RI (Table 1) were employed in the WS-CRISPR assay to reduce the reaction temperature of isothermal amplification.
UAAUUUCUACUAAGUGUAG
AUCCCUACUGCUGCCUGGA
UAAUUUCUACUAAGUGUAG
AUCAUCACCGCCAUUGCCA
UAAUUUCUACUAAGUGUAG
AUUUGCUGCUGCUUGACAG
Therefore, at least in part through supplementing PPase and employing phosphorothioated inner primers, one-pot isothermal WS-CRISPR assay was successfully developed (
Concentrations of PPase, Mg2+ ion, Bst DNA polymerase, and SuperScript IV reverse transcriptase were further evaluated. As shown in
As shown in
The digital WS-CRISPR assay is developed through partitioning the newly established one-pot WS-CRISPR reaction mixture into sub-nanoliter microreactions in the QuantStudio 3D digital chips. As shown in
Next, the effect of waiting time at room temperature on digital CRISPR assays was evaluated. Typically, it takes 5-10 min to prepare one-pot CRISPR reaction mixture. Various waiting times at room temperature during the one-pot reaction preparation and distribution steps were tested (
The specificity of the digital WS-CRISPR was tested using non-SARS-CoV-2 nucleic acids. As shown in
In order to demonstrate the feasibility for clinical SARS-CoV-2 sample testing, the digital WS-CRISPR assay was used to detect SARS-CoV-2 RNA extracted from 32 clinical swab samples and 3 clinical saliva samples. Meanwhile, an in-home RT-qPCR assay using the U.S. CDC-approved SARS-CoV-2 N1 gene's primers and probes (provided by Integrated DNA Technologies) were set up as the parallel experiment. As shown in
Saliva testing is advantageous over swab testing, since saliva samples can be self-collected by patients themselves, avoiding direct interaction between health care workers and patients (32). Given this, whether the digital WS-CRISPR assay can directly detect SARS-CoV-2 in crude saliva samples without nucleic acid extraction step was tested. As shown in
Described herein is a digital warm-start CRISPR-Cas12a (WS-CRISPR) assay for sensitive quantitation of SARS-CoV-2 from clinical samples. A one-pot warm-start CRISPR reaction combing a reverse transcription isothermal nucleic acid amplification (RT-DAMP) and CRISPR-Cas12a-based detection was used. To coordinate these two functions in one solution, pyrophosphatase and phosphorothioated inner primers were used to keep free magnesium ions stable for efficient CRISPR-Cas12a-based detection and to mediate efficient reverse transcription isothermal amplification at unfavorable temperatures such as 52° C., respectively. Through partitioning this one-pot reaction mixture into sub-nanoliter microreactions using QuantStudio 3D digital chips, the warm-start digital WS-CRISPR assay was successfully developed.
Digital WS-CRISPR assay offers several remarkable advantages. First, digital WS-CRISPR enables the successful one-pot and one-step reaction with Bst DNA polymerase-based reverse transcription isothermal amplifications (e.g., RT-DAMP and RT-LAMP) and CRISPR-Cas12a detection system, not relying on CRISPR-Cas12b which needs a relatively long crRNA (33, 34). Second, digital WS-CRISPR assay is typically initiated at a warm temperature (e.g., −50° C.) without regard to premature target amplification, an uncontrollable challenge in current digital CRISPR assays. Third, digital WS-CRISPR has high detection specificity and 10-fold higher sensitivity than tube-based bulk assay format. By targeting the SARS-CoV-2's N gene, digital WS-CRISPR is able to quantify down to 50 copies/μl SARS-CoV-2 RNA in the sample, equivalently 5 copies/μl SARS-CoV-2 RNA in the chip. Fourth, digital WS-CRISPR can quantify the viral loading in SARS-CoV-2's clinical samples, benefiting assessing the COVID-19 infectivity and the efficacy of antiviral drugs. Last, the digital WS-CRISPR assay can be used for direct saliva SARS-CoV-2 testing without time-consuming RNA extraction. This potential feature not only facilitates the COVID-19 diagnosis but also lowers the risk of infection in health workers without directly sampling from patients. In sum, the digital WS-CRISPR assay described herein provides a reliable, sensitive and straightforward platform for SARS-CoV-2 quantitative detection.
Bovine serum albumin (BSA, 20 mg/ml), EnGen Lba Cas12a (100 μM), deoxynucleotide (dNTP) mix (10 mM of each), RNase inhibitor (Murine, 40,000 U/ml), extreme thermostable single-stranded DNA binding protein (ET-SSB, 500 μg/ml), isothermal amplification buffer pack (10×; containing 200 mM Tris-HCl, 500 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Tween 20, and pH=8.8 at 25° C.), thermostable inorganic pyrophosphatase (PPase, 2,000 U/ml), Bst DNA polymerase (large fragment), Bst 2.0 DNA polymerase, Bst 3.0 DNA polymerase, and nuclease-free water were purchased from New England BioLabs (Ipswich, MA). GspSSD 2.0 DNA polymerase was from OptiGene (West Sussex, UK). IsoPol BST+ and IsoPol SD+ polymerases were purchased from ArcticZymes Technologies (Norway). Invertase from Saccharomyces cerevisiae (Grade VII, >300 U/mg), taurine (>99%, 10 g), TCEP-HCl (Reagent Grade, 5 g), and NaOH (>98%, 500 g) were purchased from Sigma-Aldrich (St. Louis, MO). UltraPure EDTA (0.5 M, pH=8), Bsm DNA Polymerase, large fragment (8 U/μl), SuperScript IV reverse transcriptase (200 U/μL), QuantStudio 3D digital PCR 20K chip kit (Version 2), and digital PCR master mix were purchased from Thermo Fisher Scientific (Waltham, MA). Normal saliva human fluid was purchased from MyBioSource (San Diego, CA). Heat-inactivated SARS-CoV-2 (Isolate USA-WA1/2020, NR-52350) was from BEI Resources (Manassas, VA). Synthetic SARS-CoV-2 RNA control (MN908947.3) with a coverage of greater than 99.9% of the bases of the SARS-CoV-2 viral genome was purchased from Twist Bioscience (San Francisco, CA). Alt-R A.s. Cas12a Ultra (500 μg; 64 μM), SARS-CoV-2 positive control (SARS-CoV-2_PC, Catalog #10006625), SARS-CoV control (Catalog #10006624), and Middle East respiratory syndrome coronavirus control (MERS-CoV control, Catalog #10006623), human RPP30 gene control (Hs_RPP30_PC, Catalog #10006626), nCOV_N1 Forward Primer Aliquot (50 nmol, Catalog #10006821), nCOV_N1 Reverse Primer Aliquot (50 nmol, Catalog #10006822), and nCOV_N1 Probe Aliquot (50 nmol, Catalog #10006823) were purchased from Integrated DNA Technologies (Coralville, IA). All clinical samples were handled in compliance with ethical regulations and the approval of Institutional Review Board of the University of Connecticut Health Center (protocol #: P61067).
Design of Primers and crRNA
Six DAMP primers and one CRISPR-Cas12a's crRNA were designed to target seven distinct sites in the 173 bp SARS-CoV-2 N gene fragment with the location from 28769 to 28941 in the viral genome (GenBank accession MW202218.1). The selected primer or crRNA recognition sites were checked to be highly conserved based on the GISAID-provided genomic epidemiology of hCoV-19 for 3564 genomes sampled between December 2019 and November 2020 (as of Nov. 11, 20202. https://www.gisaid.org/epiflu-applications/phylodynamics/). The DAMP primers can be manually designed using the OligoAnalyzer Tool (https://www.idtdna.com/pages) and the PrimerExplorer (http://primerexplorer.jp/e/) according to previously reported design principles (23). Also, they can be designed using the online DAMP primer design platform (https://github.com/xuzhiheng001/DAMP-Design). CRISPR-Cas12a's crRNA targeting the middle site of DAMP amplification region (see
The one-pot isothermal CRISPR assay system was prepared separately as Component A and B. Component A consisted of 1× isothermal amplification buffer (20 mM Tris-HCl, 50 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% (v/v) Tween 20, and pH=8.8 at 25° C.), 2 U/μl SuperScript IV reverse transcriptase, 50 mM taurine, 1 U/μl invertase, 0.01 mg/ml BSA, 0.4 mM each of dNTPs, 0.2 μM FO primer, 0.2 μM RO primer, 1.6 μM PS-FI primer, 1.6 μM PS-RI primer, 1.6 μM FC primer, 1.6 μM RC primer, 10 μM ssDNA-FQ, 20 ng/μl ET SSB, 24 U/μl Bst DNA polymerase (large fragment), 0.2 U/μl PPase, and 1 U/μl RNase inhibitor. Component B contained 1.28 μM A.s. Cas12a (Ultra) and 1.2 μM DAMP-crRNA (table 1). All the indicated concentrations were calculated based on the finally assembled reaction system. In a typical 15-1 assay, 12.5 μl Component A was first mixed with 1.5 μl of the target solution and then supplemented with 1.0 μl Component B. The assembled reaction mixture was then incubated at 52° C. for 90 min in the Bio-Rad CFX96 Touch Real-Time PCR Detection System (Hercules, CA) for real-time fluorescence detection. After incubation, the tubes were placed in the Maestrogen UltraSlim LED blue light illuminator (Pittsburgh, PA) or the Bio-Rad ChemiDoc MP Imaging System with its built-in UV channel (Hercules, CA) for endpoint visual detection. The endpoint fluorescence was the determined raw fluorescence subtracting the averaged raw fluorescence of non-template control reaction. Specificity assay was investigated by using the control plasmids containing the complete N gene from SARS-CoV-2_PC, SARS-CoV control, MERS-CoV control, and Hs_RPP30_PC. Sensitivity assay was conducted by testing serially diluted SARS-CoV-2 RNA (Twist Bioscience) in water to concentrations of 5×105, 5×104, 5×103, 5×102, and 5×101 copies/μl. CRISPR-Cas12a-based detection, namely trans-cleavage assay was incubated at 37° C. for 40 min and performed in a solution containing 1×isothermal amplification buffer, 0.32 μM A.s. Cas12a, 0.32 μM DAMP-crRNA, 1.0 μM synthetic SARS-CoV-2 N DNA fragment, and 1.0 μM ssDNA-FQ reporter.
The reaction system for digital WS-CRISPR assay was the same as that in tube-based bulk reaction mentioned above. The procedure of digital WS-CRISPR assay was modified based on the operational workflow for QuantStudio 3D digital PCR (Quick Reference Manual. Thermo Fisher Scientific). Briefly, a 15-μl digital WS-CRISPR reaction solution was first prepared in a tube by mixing Component A, B and the sample. Then, the reaction solution was loaded into the QuantStudio 3D digital PCR chip (version 2), followed by applying the lid, loading the immersion fluid, and sealing the chip. This step can be finished by using the QuantStudio 3D digital PCR Chip Loader (Thermo Fisher Scientific). Afterwards, the sealed chip was placed in ProFlex 2×Flat PCR System (Thermo Fisher Scientific) for 90-min incubation at 52° C. After incubation, the chip was taken out for the examination using a ZEISS Axio Observer fluorescence microscopy connected with ZEISS Axio Cam 305 and X-Cite 120Q fluorescence lamp illumination. For each chip's microscopy, the same parameters were set up including 5× magnification objective, 10× magnification eyepiece, 700 ms exposure time, 2.1 gamma value, and 2000 white value. Six distinct regions without overlapping areas were randomly captured by the microscopy to cover about 2809 microreactions. The number of positive spots was counted by using the ImageJ software. The step-by-step clicking after opening the images is Image>Type (8-bit)>Edit>Invert>Image>Adjust (Threshold: 0 and 245)>Apply>Analyze>Analyze Particles>Distribution>List. In the list, the count for over 0.02 bin start was enrolled and summed up.
Similarly, the specificity assay for the digital WS-CRISPR was finished by testing the control plasmids mentioned above and sensitivity was evaluated by testing serially diluted SARS-CoV-2 RNA (Twist Bioscience) in water to concentrations of 5×105, 5×104, 5×103, 5×102, 5×101 and 5×100 copies/μl, as well as 3×106 copies/μl SARS-CoV-2 RNA extracted from a saliva sample. SARS-CoV-2 in each clinical sample was quantified using the calibration curve of digital WS-CRISPR by applying the averaged percentage of positive spots in six micrographs. Direct saliva digital WS-CRISPR assays were assessed by testing five saliva mock samples. The reaction system and procedure were the same as described above, replacing the target solution with 1.5 μl of heat-treated saliva mock sample solution at 95° C. for 5 min. The saliva mock samples (400 μl) were prepared by adding 1× inactivation reagent (0.0115 N NaOH, 1.0 mM EDTA, and 1.0 mM TCEP-HCl), 360 μl human saliva, and various volume percentages (0%, 1%, 2.5%, 5%, and 10%) of heat-inactivated SARS-CoV-2.
The real-time quantitation RT-PCR (RT-qCPR) assay for SARS-CoV-2 detection was carried out according to U.S. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (https://www.fda.gov/media/134922/download) with minor modifications. A typical RT-qPCR reaction system included 2 U/μl SuperScript IV reverse transcriptase, 1× QuantStudio master mix (Catalog #A26358), 0.5 μM nCOV_N1 forward primer, 0.5 μM nCOV_N1 reverse primer, 0.125 μM nCOV_N1 probe, and 1.5 μl of the target solution. The thermal cycler protocol consisted of Stage 1 (2.0 min at 25° C.), Stage 2 (15.0 min at 50° C.), Stage 3 (2.0 min at 95° C.) and Stage 4 (40 cycles of 3.0 s at 95° C. and 30 s at 55° C.). The capture point of fluorescence was set at 55° C. in Stage 4. Real-time quantitation analysis was performed in the Bio-Rad CFX96 Touch Real-Time PCR Detection System (Hercules, CA). Through testing serially diluted SARS-CoV-2 RNA (Twist Bioscience) with concentrations of 5×105, 5×104, 5×103, and 5×102 copies/μl (
GraphPad Software Prism 8.0.1 was used to plot real-time fluorescence curves, analyze linear regression, and verify statistical significances between two assay groups. The unpaired two-tailed t-test was made with the p value <0.05 as the threshold for defining significance. For endpoint imaging of the chip using fluorescence microscopy, six distinct regions without overlapping areas were randomly taken to cover about 2809 microreactions. Unless otherwise specified, each image for visual detection or micrograph for chip testing is a representative of at least three independent experiments. To plot the linear relationship between percentage of positive spots and concentration of targets in digital WS-CRISPR, total positive spots in all the six micrographs were used and three chips were taken to run three independent assays. Clinical sample testing by both digital WS-CRISPR and RT-qPCR assays was repeated three times to ensure data accuracy.
This application claims priority to U.S. Provisional Patent Application No. 63/115,291, filed Nov. 18, 2020, the entire contents of which are incorporated herein by reference for all purposes.
This invention was made with government support under Grant No. R01EB023607, R61AI154642, and R01CA214072 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/059727 | 11/17/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63115291 | Nov 2020 | US |
