ENGINEERED CRISPR-CAS SYSTEMS AND METHODS FOR SENSITIVE AND SPECIFIC DIAGNOSTICS
20230057482 · 2023-02-23
Inventors
- Blake A. WIEDENHEFT (Bozeman, MT, US)
- Andrew SANTIAGO-FRANGOS (Bozeman, MT, US)
- Anna A. NEMUDRAIA (Bozeman, MT, US)
- Artem A. NEMUDRYI (Bozeman, MT, US)
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
C12Q1/6818
CHEMISTRY; METALLURGY
C12Q1/6818
CHEMISTRY; METALLURGY
C12N2800/80
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
International classification
C12N15/11
CHEMISTRY; METALLURGY
Abstract
The disclosure relates to an engineered type III CRISPR-Cas system for sensitive and sequence specific detection of nucleic acid in a sample. For example, the engineered type III CRISPR-Cas system may be implemented as an assay for testing SARS-CoV-2 virus (or other target nucleic acid in the sample) that can be performed quickly, such as in one hour or less. Nucleic acid recognition by type III systems may trigger Cas10-mediated nuclease activity and/or polymerase activity, which may generate pyrophosphates, protons and cyclic oligonucleotides. The nuclease activity and/or the one or more products of the Cas10-polymerase are detected using colorimetric, visible fluorometric, and/or instrumented fluorometric detection.
Claims
1. A method of detecting nucleic acid in a sample, comprising: contacting the sample with an engineered type III Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system to detect NA in the sample, the engineered CRISPR-Cas system comprising: a CRISPR guide comprising a CRISPR guide sequence that is complementary to a locus of the nucleic acid; a first subunit that undergoes a conformational change upon binding of the engineered type III CRISPR-Cas system to the locus of the nucleic acid, the conformational change activating DNase activity of the first subunit and/or polymerase activity of the first subunit, the polymerase activity generating one or more products; and detecting the DNase activity and/or the one or more products of the polymerase activity.
2. The method of claim 1, wherein the nucleic acid comprises a viral ribonucleic acid (RNA).
3. The method of claim 2, wherein the viral RNA comprises RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
4. The method of claim 3, wherein the locus comprises a nucleocapsid gene (N-gene) of the SARS-CoV-2.
5. The method of claim 3, wherein the locus comprises a region of the viral RNA that is conserved among a plurality of SARS-CoV-2 genomes.
6. The method of claim 2, wherein the CRISPR guide sequence comprises a nucleic acid sequence of SEQ ID NO. 1.
7. The method of claim 2, wherein the CRISPR guide sequence comprises a nucleic acid sequence of SEQ ID NO. 2.
8. The method of claim 1, wherein the one or more products comprise a linear or cyclic oligonucleotide and wherein the detection system comprises instrumented fluorometric detection comprising: an RNA tether linking a fluorophore to a quencher; and a nuclease activated by the linear or cyclic oligonucleotide, the activated nuclease cleaving the RNA tether to thereby release the fluorophore that is detected by a fluorescence detecting instrument.
9. The method of claim 8, wherein the linear or cyclic oligonucleotide comprises a cyclic oligoadenylate, and wherein the nuclease activated by the linear or cyclic oligonucleotide comprises Csm6 or Can1.
10. The method of claim 8, wherein the instrumented fluorometric detection further comprises: a deoxyribonucleic acid (DNA) tether linking the fluorophore or a second fluorophore to the quencher or a second quencher, wherein the DNase activity cleaves the DNA tether to thereby release the fluorophore or the second fluorophore.
11. The method of claim 1, wherein the detection system comprises instrumented fluorometric detection comprising: a deoxyribonucleic acid (DNA) tether linking a fluorophore to a quencher, wherein the first subunit has a DNase activity that is activated upon hybridization of the RNA guide to the locus of the viral RNA, the DNase activity cleaving the DNA tether to thereby release the fluorophore that is detected.
12. The method of claim 1, wherein the one or more products comprise a linear or cyclic oligonucleotide and wherein the detection system comprises instrumented fluorometric detection comprising: a deoxyribonucleic acid (DNA) tether linking a fluorophore to a quencher; and a nuclease activated by the linear or cyclic oligonucleotide, the activated nuclease cleaving the DNA tether to thereby release the fluorophore that is detected by a fluorescence detecting instrument.
13. The method of claim 1, wherein the one or more products comprise a pyrophosphate, and wherein the detection system comprises visible fluorometric detection comprising: a fluorescent dye quenched by a quencher; wherein the pyrophosphate forms an insoluble precipitate with the quencher to thereby unquench the fluorescent dye that is detected based on a color change.
14. The method of claim 13, wherein the fluorescent dye comprises calcein and the quencher comprises Manganese, and wherein unquenched calcein is bound by Magnesium to form a fluorescent complex that is detected.
15. The method of claim 1, wherein the one or more products comprise protons, and wherein the detection system comprises a colorimetric system, the colorimetric system comprising: a solution comprising a pH-sensitive dye; and wherein the protons acidify the solution, resulting in a change in color of the pH-sensitive dye.
16. The method of claim 1, wherein the engineered type III CRISPR-Cas system further comprises: an engineered second subunit comprising a backbone subunit of the engineered type III CRISPR-Cas system with an introduced mutation, the engineered second subunit having RNase activity when in wildtype form, but the introduced mutation disrupting the RNase activity to prevent degradation of the viral RNA, thereby increasing signal generation of the detection system.
17. The method of claim 13, wherein the first subunit comprises a Cas10 subunit, the second subunit comprises Csm3, and wherein an activity of the Cas10 subunit is moderated by activity of the second subunit in the wildtype form, and wherein the introduced mutation to the second subunit disrupts the moderation of the Cas10 subunit.
18. The method of claim 16, wherein the wildtype form of the second subunit comprises an amino acid sequence of SEQ ID NO. 26 and the second subunit with the introduced mutation comprises an amino acid sequence of SEQ ID NO. 27.
19. The method of claim 1, wherein the one or more products comprise (i) a linear or cyclic oligonucleotide and (ii) protons, wherein the detection system comprises: fluorometric detection comprising: an RNA tether linking a fluorophore to a quencher; a nuclease activated by the linear or cyclic oligonucleotide, the activated nuclease cleaving the RNA tether to thereby release the fluorophore that is detected; and colorimetric detection comprising: a solution comprising a pH-sensitive dye; and wherein the solution is acidified by the protons resulting in a change in color of the pH-sensitive dye.
20. The method of claim 19, wherein the fluorometric detection further comprises: a deoxyribonucleic acid (DNA) tether linking the fluorophore or a second fluorophore to the quencher or a second quencher, wherein the DNase activity cleaves the DNA tether to thereby release the fluorophore or the second fluorophore.
21. The method of claim 1, wherein the one or more products comprise protons, wherein the detection system comprises: fluorometric detection comprising: a deoxyribonucleic acid (DNA) tether linking a fluorophore to a quencher, wherein the DNase activity cleaves the DNA tether to thereby release the fluorophore that is detected; and colorimetric detection comprising: a solution comprising a pH-sensitive dye; and wherein the solution is acidified by the protons resulting in a change in color of the pH-sensitive dye.
22. The method of claim 1, wherein the nucleic acid comprises ribonucleic acid (RNA), the system further comprising: a reverse transcription loop-mediated isothermal amplification (RT-LAMP) primer having a T7 binding site for RT-LAMP-T7 amplification of the RNA.
23. The method of claim 22, wherein the RT-LAMP-T7 amplification and the detection of the RNA comprises a single pot combination.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION
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[0046] The engineered type III CRISPR-Cas system may be implemented as an assay for testing SARS-CoV-2 virus (or other target nucleic acid in the sample) that can be performed quickly, such as in one hour or less. Nucleic acid recognition by type III systems may trigger Cas10-mediated nuclease activity and/or polymerase activity, which may generate one or more products such as pyrophosphates, protons and cyclic oligonucleotides. The nuclease activity and/or the one or more products of the Cas10-polymerase are detected using colorimetric, visible fluorometric, and/or instrumented fluorometric detection.
[0047] The engineered system 100 may include a modified CRISPR complex, detection components, and/or other components. The modified CRISPR complex may include a modified type III CRISPR complex. The modified CRISPR complex may include a CRISPR guide and a plurality of subunits.
[0048] The plurality of subunits may include a CRISPR guide, Cas10 subunit, backbone subunits associated with the Csm (such as Csm3) or Cmr systems, and/or other subunits necessary for assembly of the type III surveillance complex as well as the ancillary nucleases (such as Csm6, Can1, Csx). Various examples described herein may describe a CRISPR complex purified from the organism Thermus thermophilus. These examples may further describe the use of protein subunits of T. thermophilus CRISPR complexes. Accordingly, these examples may refer to the subunits as TtCas10, TtCsm3, TtCsm6, and so forth. It should be noted, however, that other protein subunits that perform similar functions may be used as well and/or instead of these examples of subunits.
[0049] The CRISPR guide may include a CRISPR guide sequence that is engineered to be complementary to a locus of the nucleic acid. The CRISPR guide sequence may be selected based on one or more conserved regions of the target nucleic acid. For example,
[0050] The CRISPR guide sequence may be designed based on conserved sequence across different samples of the SARS-CoV-2, different strains of the SARS-CoV-2, and/or other samples available for the SARS-CoV-2. Such conserved sequence may be determined based on sequence alignments. A pairwise match may be considered when an alignment quality of the pairwise match is sufficient to determine that aligned portions of two sequences represent a conservation of the nucleotides in the sequences across genomes of SARS-CoV-2 (or other target). The alignment quality may be specified as having a minimum overlap of at least about 1 base, 2 bases, 4 bases, 4 bases, 5 bases, 10 bases, 15 bases, 40 bases, 25 bases, 40 bases, 45 bases, 40 bases, 45 bases, 50 bases, 55 bases, 60 bases, 65 bases, 70 bases, 75 bases, 80 bases, 85 bases, 90 bases, 95 bases, or 100 bases. Alternatively, or additionally, the alignment quality may be based on a minimum alignment identity of at least about 5%, 10%, 15%, 40%, 25%, 40%, 45%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some cases, a criterion may require at least a 25-nt overlap with at least about 70% identity.
[0051] In this example, the sequencing encoding the SARS-CoV-2 nucleocapsid (N) gene was selected to serve as the basis for generating the CRISPR guide sequence. Examples of the CRISPR guide sequence include SEQ ID NO. 1 and SEQ ID NO. 2.
[0052] Recently, loop-mediated isothermal amplification (LAMP) (Notomi et al., 2000) has been developed as a sensitive (1-100 copies/μL) point-of-care diagnostic (Dao Thi et al., 2020; Zhang et al., 2020). However, LAMP is prone to generating false positives unless a second sequence-specific technique is used to check the amplified DNA (Dao Thi et al., 2020; Rolando et al., 2020). The type V (Cas12-based) and type VI (Cas13-based) CRISPR systems have been coupled to LAMP or RPA (recombinase polymerase amplification) for sensitive and reliable detection of viral nucleic acids (Broughton et al., 2020; Chen et al., 2018; Gootenberg et al., 2018, 2017; Joung et al., 2020). Following isothermal amplification, the RNA-guided Cas12 or Cas13 proteins bind to the amplified target and trigger a non-sequence specific nuclease activity that cleaves a fluorophore and quencher labelled DNA or RNA (Chen et al., 2018; Gootenberg et al., 2018). Cleavage of the tether results in an increase in fluorescence that can be detected in 45 minutes. While Cas12 and Cas13 detection methods have been optimized over several iterations to be compatible with isothermal amplification of viral RNA, the ultimate goal is to develop CRISPR-based technologies that are sensitive enough to detect the viral RNA directly, without prior amplification. Recently, Fozouni et al reported that the type IV (Cas13a-based) CRISPR systems can be used for amplification-free detection of SARS-CoV-2 RNA in ˜30 minutes and with sensitivity of ˜100 copies/μL (Fozouni et al., 2020).
[0053] Like the type VI (Cas13-based) systems, type III systems also target RNA (Hale et al., 2009; Kazlauskiene et al., 2016; Samai et al., 2015; Staals et al., 2014; Tamulaitis et al., 2014). However, type III systems rely on a unique intrinsic signal amplification mechanism (
[0054] Results and Discussion
[0055] Sequence-Specific Activation of Cas10 Polymerase Yields Three Detectable Products
[0056] Sequence-specific recognition of RNA by type III CRISPR systems initiates a signaling Cascade, as illustrated in
[0057] Still referring to
[0058] An example of purification of TtCsm complexes and TtCsm6 is illustrated in
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[0064] Referring to
TABLE-US-00001 TABLE 1 Examples of fluorescent reporter RNAs. Name Sequence (5’-3’) RNA reporter A /56-FAM/rCrUrCrUrCrU/3IABkFQ/(FIG. 1) RNA reporter B /56-FAM/rArUrCrUrUrCrUrUrArU/3IABkFQ/ (FIGS. 2 and 3)
[0065] Single mismatches in the target RNA have been shown to result in 10-fold lower amounts of cyclic oligoadenylate production by other Csm complexes (Nasef et al., 2019). Using fluorometric detection, both the mutant and the wildtype Csm complex could detect the SARS-CoV-2 RNA at concentrations above 10.sup.8 copies per reaction, and neither complex cross-reacted with the SARS-CoV-1 RNA at the highest concentrations tested. The RNase-dead TtCsm complex was roughly 3-fold more sensitive than wildtype, with an LoD of ˜10.sup.7 copies per reaction.
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[0067] Csm-Based Direct Detection of SARS-CoV-2 RNA in Patient Samples
[0068] The LoD using crRNA.sub.N1 is between 10.sup.7 and 10.sup.8 copies of IVT RNA per μL, which is insufficient to be clinically relevant. To identify other guides that might outperform or complement the activity of crRNA.sub.N1 we aligned 45,641 SARS-CoV-2 genomes available from GISAID (Elbe and Buckland-Merrett, 2017). These alignments were used to select guides based on four key criteria. First, each target sequence had to be more than 99% identical among the available SARS-CoV-2 genomes. Second, complementarity between the target and the crRNA was not allowed to extend beyond the spacer sequence (guide), and into repeat derived portions of the crRNA that have been shown to suppress Cas10 activity (Kazlauskiene et al., 2017). Third, we targeted regions of SARS-CoV-2 that were different by at least two-nucleotides in SARS-CoV-1 and MERS-CoV. Fourth, the list of target sequences was pruned to remove guides with similarity to human mRNA sequences, or common oral and respiratory pathogen sequences (E-value <1000). Finally, we focused on target sequences located 3′ of the ORF3a gene, which are present on both the viral genome and on subgenomic RNAs generated during infection. In total, we designed crRNAs targeting 10 different locations on the SARS-CoV-2 genome as illustrated in
[0069] To determine how each of these guides perform, we measured sequence specific detection of RNA using a fluorometric reporter assay (i.e., FAM-RNA-Iowa Black FQ), results of which are illustrated in
[0070] Fozouni et al., recently showed that multiplexing Cas13 (i.e., combining multiple guides into a single reaction) improves the sensitivity of SARS CoV-2 detection (Fozouni et al., 2020). We reasoned that similar benefits might be possible for Csm-based detection. To test this idea, we combined 10 of the guides (2.5 nM each) into a single multiplexed reaction. Multiplexing 10 guides improves the sensitivity of TtCsm-mediated detection of SARS-CoV-2 RNA isolated form the nasal swab of a positive patient by approximately 10 times as shown in
[0071] Testing Clinical Samples for SARS-CoV-2 Using RT-LAMP and T7-Csm.
[0072] Csm-based detection is currently not sensitive enough to directly detect SARS-CoV-2 in all patients capable of spreading the infection, which requires an LoD of 10.sup.3 RNA copies/μL. (La Scola et al., 2020; Larremore et al., 2021; Paltiel et al., 2020; Wölfel et al., 2020). To decrease the LoD of a type III CRISPR-based diagnostic to 10.sup.3 RNA copies/μL or lower, we incorporated an upstream nucleic acid amplification technique as illustrated in
[0073] To confirm the specificity of TtCsm-based detection, we tested SARS-CoV-2 alongside a panel of eight other oral and respiratory pathogens, including coronaviruses SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), Human coronavirus HKU1 and Human coronavirus NL63 as illustrated in
[0074] These samples resulted in background signal similar to the no template control (NTC). In contrast, SARS-CoV-2 RNA results in a 4-5-fold increase in signal.
[0075] To determine the LoD of RT-LAMP-T7-Csm, we tested 20 replicates of 2-fold serial dilutions ranging from ˜100-400 copies/μL SARS-CoV-2 RNA as illustrated in
[0076] The LoD of RT-LAMP-T7-Csm is 198 copies/μL SARS-CoV-2 RNA (20/20 replicates), in an assay that relies on a 29-minute RT-LAMP step, followed by a 1-minute T7-Csm fluorometric detection reaction.
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[0078] To further validate this method, we next tested RNA extracted from 56 nasopharyngeal swab samples taken from patients that had previously been tested using RT-qPCR. Of the 56 samples tested, 46 were positive for SARS-CoV-2 and 10 were negative by RT-qPCR as illustrated in
[0079] Using two different crRNA guides, we demonstrate that the type III CRISPR system has a specificity (negative predictive agreement) of 100%, as well as a positive predictive agreement of 100% for nasopharyngeal swab samples with 100-200 copies/μL SARS-CoV-2 RNA as determined by RT-qPCR. Whole genome sequencing revealed three of the patient samples used here belonging to the B.1.1.7. lineage. These genome sequences have been deposited in GISAID (Accession IDs: EPI_ISL_1081321, EPI-ISL_1081322, EPI_ISL_1081323) (Elbe and Buckland-Merrett, 2017). Importantly, the B.1.1.7. variants were positively identified by RT-LAMP-T7-Csm with both N1 and N9 crRNA guides (illustrated in
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[0082] In some examples, at least one of the plurality of subunits may be genetically modified. For example, the TtCsm3 subunit may be genetically modified according to the sequence of SEQ ID NO. 27.
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[0086] Examples of Methods
[0087] Nucleic Acid Preparation
[0088] Previously published LAMP primers (Eurofins) were designed to amplify the SARS-CoV-2 N-gene (Broughton et al., 2020). Target SARS-CoV-2 and SARS-CoV-1 RNAs were in vitro transcribed with MEGAscript T7 (Thermo Fisher Scientific) from PCR products generated from pairs of synthesized overlapping DNA oligos or using SARS-CoV-2 genome as a template (SEQ ID NOS. 13-17). Previously designed primer pools (IDT) were used for RT-PCR and sequencing of SARS-CoV-2 genomes (https://artic.network/ncov-2019) (link should omit spaces). Transcribed RNAs were purified by denaturing PAGE. Fluorescent reporter RNA A and fluorescent reporter RNA B purified by RNase-free HPLC (See Table 1) (IDT). Purified genomes of viral, bacterial and fungal pathogens were used as is, or resuspended in 1×TE (10 mM Tris-HCl pH 7.5, 1 mM Ethylenediaminetetraacetic acid (EDTA)) to ˜1×10.sup.6 genomes/μL. Examples of purified genomic nucleic acids (such as purified genomes of the viral, bacterial and fungal pathogens) are illustrated in Table 2.
TABLE-US-00002 Name Source SARS-CoV-2 The National Institute of Standards and Technology (RGTM 10169) SARS-CoV-1 American Type Culture Collection (ATCC) (VR-3280SD) MERS-CoV ATCC (VR-3248SD) Human coronavirus ATCC (VR-3262SD) HKU1 Influenza B ATCC (VR-1885DQ) Human coronavirus ATCC (VR-3263SD) NL63 Human respiratory ATCC (VR-1580DQ) syncytial virus Pseudomonas ATCC (27853D-5) aeruginosa Candida albicans ATCC (10231D-5)
[0089] Plasmids
[0090] Expression vectors for Thermus thermophilus type III-A Csm1-Csm5 genes, pCDF-5×T7-TtCsm (Liu et al., 2019) were used as a template for site-directed mutagenesis to mutate the Csm3 residue D33 to alanine (D33A) to inactivate Csm3-mediated cleavage of target RNA (pCDF-5×T7-Tt.sup.CsmCsm3-D34A) (Liu et al., 2017). The CRISPR array in pACYC-TtCas6-4×crRNA4.5 (Liu et al., 2019) was replaced with a synthetic CRISPR array (GeneArt) containing five repeats and four identical spacers, designed to target the N-gene of SARS-CoV2 (i.e., pACYC-TtCas6-4×gCoV2N1). TtCas6 was PCR amplified from the pACYC-TtCas6-4×crRNA4.5 plasmid and cloned between the NcoI and XhoI sites of pRSF-1b (pRSF-TtCas6). The CARF-HEPN nuclease TtCsm6 was expressed from pC0075 TtCsm6 His6-TwinStrep-SUMO-BsaI (Gootenberg et al., 2018).
[0091] Protein Purifications
[0092] Expression and purification of the TtCsm complex was performed as previously described with minor modifications (Liu et al., 2019). Briefly, the crRNA plasmid (such as pACYC-TtCas6-4×gCoV2N1) was co-transformed with pRSF-TtCas6 and either pCDF-5×T7-TtCsm or pCDF-5×T7-Tt.sup.CsmCsm3-D34A into Escherichia coli BL21(DE3) cells and grown in LB Broth (Lennox) (Thermo Fisher Scientific) at 37° C. to an OD.sub.600 of 0.5. Cultures were then induced with 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) for expression overnight at 25° C. Cells were pelleted (3,000×g for 25 mins at 4° C.) and lysed via sonication in Lysis buffer (25 mM HEPES pH 7.5, 150 mM KCl, 10 mM imidazole, 1 mM TCEP, 0.01% Triton X-100, 5% glycerol, 1 mM PMSF). Lysate was clarified by centrifugation at 10,000×g for 25 mins at 4° C. The lysate was then heat-treated at 55° C. for 45 minutes and further clarified by centrifugation at 10,000×g for 25 mins at 4° C. His-tagged Csm1 and TtCsm complex were bound to HisTrap HP resin (Cytiva) and washed with Wash buffer (50 mM HEPES pH 7.5, 150 mM KCl, 1 mM TCEP, 5% glycerol, 20 mM imidazole). Protein was eluted in Lysis buffer supplemented with 300 mM imidazole. Eluted protein was concentrated (100 k MWCO Corning Spin-X concentrators) at 4° C. before further purification over HiLoad Superdex 200 26/600 or Superose 6 Increase 10/300 GL size-exclusion columns (Cytiva) in 25 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM TCEP. Fractions containing the TtCsm complex were pooled, concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at −80° C.
[0093] Expression and purification of TtCsm6 was performed as previously described with minor modifications (Gootenberg et al., 2018). pTtCsm6 was transformed into Escherichia coli BL21(DE3) cells and grown in LB Broth (Lennox) (Thermo Fisher Scientific) at 37° C. to an OD.sub.600 of 0.5. Cultures were then incubated on ice for 1 hour, and then induced with 0.5 mM IPTG for expression overnight at 16° C. Cells were lysed via sonication in TtCsm6 Lysis buffer (20 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM TCEP) and lysate was clarified by centrifugation at 10,000×g for 25 mins at 4° C. The lysate was heat-treated at 55° C. for 45 minutes and clarified by centrifugation at 10,000×g for 25 mins at 4° C. His6-TwinStrep-tagged TtCsm6 was bound to StrepTrap HP resin (Cytiva) and washed in TtCsm6 Lysis buffer. The protein was eluted with TtCsm6 Lysis buffer supplemented with 2.5 mM desthiobiotin and concentrated (10 k MWCO Corning Spin-X concentrators) at 4° C. Affinity tags were removed from TtCsm6 using SUMO protease (100 μL of 2.5 mg/ml protease per 20 mg of TtCsm6 substrate) during dialysis against SUMO digest buffer (30 mM Tris-HCl pH 8, 500 mM NaCl 1 mM DTT, 0.15% Igepal) at 4° C. overnight. Cleaved His6-TwinStrep tag and uncleaved His6-TwinStrep-TtCsm6 were removed by binding to HisTrap HP resin (Cytiva), and the flow-through was concentrated using Corning Spin-X concentrators at 4° C. Finally, TtCsm6 was purified using a HiLoad Superdex 200 26/600 size-exclusion column (Cytiva) in 20 mM Tris-HCl pH 7.5, 1 mM DTT, 400 mM monopotassium glutamate, 5% glycerol. Fractions containing TtCsm6 were pooled, concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at −80° C.
[0094] To screen guide RNAs in a high throughput format, ten TtCsm complexes were first crudely purified. 8 mL cultures of E. coli BL21-DE3 cells transformed with pTtCsm and pT7-5×CRISPR-Cas6 were grown at 37° C. and 250 RPM in LB media with selective antibiotics until they reached an OD.sub.600 reading of 0.4. Protein expression was then induced with the addition of 0.5 mM IPTG to the media, and cells were grown overnight at 16° C. Cells were collected by centrifugation at 4000 RPM, and cell pellets were resuspended in 250 μL of Ni-NTA Equilibration buffer (PBS; 100 mM sodium phosphate, 600 mM sodium chloride), 0.05% Tween™-20 Detergent, 30 mM imidazole; pH 8.0). Resuspended cells were sonicated twice for twenty seconds, then clarified by centrifugation at 15,000 rpm for 20 minutes at −4° C. to remove cellular debris. The lysate was then heat-treated at 55° C. for 45 minutes, and re-clarified by centrifugation at 15,000 rpm, for 30 mins at 4° C. TtCsm was then purified using HisPur Ni-NTA magnetic beads (ThermoFisher) according to the manufacturers recommendations, but with modified wash (25 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween-20, 1 mM TCEP) and equilibration (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) and elution buffers (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 300 mM Imidazole). TtCsm complex concentration was quantified on a Nanodrop (ThermoFisher).
[0095] Type III CRISPR-Based RNA Detection
[0096] Fluorescent CRISPR-Csm Based Detection
[0097] For experiments shown in
[0098] For experiments shown in
[0099] Colorimetric CRISPR-Csm Based Detection
[0100] TtCsm.sup.Csm3-D34A stocks were buffer exchanged into a low buffering capacity buffer (0.5 mM Tris-HCl pH 8.8, 50 mM Potassium chloride, 10 mM Ammonium sulphate, 8 mM Magnesium sulphate) using Microspin G25 columns (Cytiva) as per the manufacturer's instructions. TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) or in vitro transcribed SARS-CoV-2 or SARS-CoV-1 RNA were incubated with 200 nM TtCsm.sup.Csm3-D34A in 1× WarmStart Colorimetric LAMP Master Mix (NEB), supplemented with an additional 1 mM ATP, in a 25 μL reaction. The volume of buffer-exchanged TtCsm used contributed approximately 40 μM Tris-HCl pH 8.8 buffer to the final reaction. Reactions were assembled on ice and imaged on an LED tracing pad with a Galaxy S9 phone (Samsung). Then reactions were incubated at 60° C. for 30 minutes, rapidly cooled, and imaged again.
[0101] Visible Fluorometric CRISPR-Csm Based Detection
[0102] TE buffer or in vitro transcribed SARS-CoV-2 or SARS-CoV-1 RNA were incubated with 500 nM TtCsm.sup.Csm3-D34A in reaction buffer (20 mM Tris-HCl pH 8.8, 100 mM Potassium chloride, 10 mM Ammonium sulphate, 6 mM Magnesium sulphate, 0.5 mM Manganese chloride, 1 mM TCEP, 1 mM ATP and 25 μM Calcein), in a 30 μL reaction. Reactions were incubated at 60° C., and fluorescence was measured over time in an ABI 7500 Fast Real-Time PCR System (Applied Biosystems), using the manufacturers default filter settings for FAM dye. After incubating at 60° C. for 50 minutes, the same reactions were then imaged under visible light, and under UV light (365 nm) with a Galaxy S9 phone (Samsung). To screen guide RNAs in a high throughput format (
[0103] RT-LAMP-T7-Csm
[0104] Isothermal amplification of nucleic acids in swab samples was performed by RT-LAMP. In brief, 25 μL reactions contained 8 units (U) of WarmStart Bst 2.0 (NEB), and 7.5 U of WarmStart RTx Reverse Transriptase (NEB), 1.4 mM dNTPs, LAMP primers, 25 U of Murine RNase Inhibitor (NEB) in reaction buffer (20 mM Tris-HCl pH 7.8, 8 mM Magnesium sulfate, 10 mM Ammonium sulfate, 50 mM potassium chloride, 0.1% Tween-20). LAMP primers designed to amplify the SARS-CoV-2 N-gene (Broughton et al., 2020), were added at an optimized final concentration of 0.2 μM F3 and B3, 0.4 μM LoopF and LoopB, 1.6 μM BIP, 0.53 μM FIP, and 1.07 μM of T7-FIP (such as the primers for RT-LAMP: SEQ ID NOS. 19-25). The T7-FIP primer consists of a T7 promoter fused to the 5′ end of the FIP primer, and allows for the generation of T7 transcription templates during the second step of T7-Csm reaction. RT-LAMP reactions were performed using 5 μL of input RNA at 65° C. for 29 minutes. 3 μL of RT-LAMP reactions were mixed with 27 μL of a modified T7-Csm fluorescent detection reaction containing 0.5 mM rNTPs, 300 nM TtCsm6, 150 nM fluorescent reporter RNA B, and 20 nM of either TtCsm.sup.Csm3-D34A N1 or N9, in reaction buffer (40 mM Tris-HCl pH 7.5, 4 mM Magnesium chloride, 50 mM Sodium chloride, 2 mM spermidine, 1 mM DTT). Reactions were incubated at 55° C. for up to 20 min and fluorescence kinetics was monitored in a QuantStudio 3 Real-Time PCR system (ThermoFisher) as described above.
[0105] LoD standards were prepared by diluting SARS-CoV-2 RNA into RNA extracted from COVID-19-negative patient nasopharyngeal swabs. Concentrations were determined with RT-qPCR using a standard curve generated from 10-fold dilution series)(1×10.sup.6-1×10.sup.0 of IVT fragment.
[0106] Human Clinical Sample Collection and Preparation
[0107] Nasopharyngeal swabs from patients that either tested negative or positive for SARS-CoV-2 were collected in viral transport media. RNA was extracted from all patient samples using QIAamp Viral RNA Mini Kit (Qiagen).
[0108] RT-qPCR
[0109] RT-qPCR was performed using two primers pairs (N1 and N2) and probes from the 2019-nCoV CDC EUA Kit (IDT #10006606). SARS-CoV-2 in RNA-extracted, nasopharyngeal patient samples was detected and quantified using one-step RT-qPCR in ABI 7500 Fast Real-Time PCR System according to CDC guidelines and protocols (https://www.fda.gov/media/134922/download) (link should omit spaces). In brief, 20 μL reactions included 8.5 μL of Nuclease-free Water, 1.5 μL of Primer and Probe mix (IDT, 10006713), 5 μL of TaqPath 1-Step RT-qPCR Master Mix (ThermoFisher, A15299) and 5 μL of the template. Nuclease-free water was used as negative template control (NTC). Amplification was performed as follows: 25° C. for 2 min, 50° C. for 15 min, 95° C. for 2 min followed by 45 cycles of 95° C. for 3 s and 55° C. for 30 s. To quantify viral genome copy numbers in the samples, standard curves for N1 and N2 were generated using a dilution series of a SARS-CoV-2 synthetic RNA fragment (RTGM 10169, National Institute of Standards and Technology) spanning N gene with concentrations ranging from 10 to 10.sup.6 copies per μL. Three technical replicates were performed at each dilution. The NTC showed no amplification throughout the 45 cycles of qPCR.
[0110] Bioinformatic Design of TtCsm crRNA Guides Targeting SARS-CoV-2
[0111] An alignment of 45,641 SARS-CoV-2 genomes was downloaded front the GISAID database (Global Initiative for Sharing All Influenza Data; GISAID.org) (link should omit spaces) on 6-23-2020 (Elbe and Buckland-Merrett, 2017; Katoh and Standley, 2013). The alignment was scanned for conservation with a 40-nucleotide sliding window, and 40-nucleotide segments with strong conservation were saved for downstream analysis. Next, four nucleotides flanking the above 40-nucleotide candidate viral target sequences were checked for base pairing to the first four nucleotides of the prospective 5′-crRNA handle (underlined; 5′-AUUGCGAC-3′), only candidates lacking handle complementarity were considered further. Candidate sites with less than two mismatches to SARS-CoV (NC_004718.3) and MERS-CoV (NC_019843.3) in the first 18 nucleotides of the target sequence were discarded. Next, candidate crRNAs targeting the above sites were screened for potential cross-reactivity with human mRNAs and a list of human pathogens and common respiratory flora downloaded from the FDA's Emergency Use Authorization requirements (downloaded on 7-29-2020) using BLAST (−evalue 1000). The remaining 6,229 crRNA sequences were then sorted by genomic location and only guides that were located 3′ of the SARS-CoV-2 ORF3a gene (positions 25,393 to 29,903) were considered further. Finally, 76 guides were selected from the remaining pool that had the greatest conservation amongst SARS-CoV-2. sequences and the largest number of mismatches to SARS and MERS-CoV sequences.
[0112] Sequencing of SARS-CoV-2 RNA Isolated from Patient Samples
[0113] SARS-CoV-2 genomic RNA isolated from patient samples was sequenced as previously described (Nemudryi et al., 2021). In brief, 10 μL of SARS-CoV-2 genomic RNA extracted from nasopharyngeal patient swabs was first reverse transcribed with SuperScript IV (ThermoFisher) according to the manufacturer's instructions. The ARTIC Network protocol was followed to generate a sequence amplicon library covering the whole SARS-CoV-2 genome on Oxford Nanopore using a ligation sequencing kit (Oxford Nanopore, SQK-LSK109) (https://artic.network/ncov-2019) (Grubaugh et al., 2019; Tyson et al., 2020) (link should omit spaces). Two multiplex PCR reactions were performed with primer pools described in the ARTIC nCoV-2019 V3 Panel (such as primers to generate an amplicon library for SARS-CoV-2 whole genome sequencing: SEQ ID NOS. 29-246), amplified with Q5 DNA Polymerase (NEB). The two resulting amplicon pools for each patient sample were then combined and used for library preparation. Samples were end repaired (NEB, E7546) and then barcoded using Native Barcoding Expansion Kits (Oxford Nanopore, EXP-NBD104 and EXP-NBD114). Barcoded samples were pooled together and then Nanopore adaptors were ligated.
[0114] The multiplexed library was loaded onto the MinION flowcell, and a total of 0.3 Gb of raw sequencing data was collected per patient sample. Raw Nanopore reads were base-called in high-accuracy mode (Oxford Nanopore, MinKNOW), and further analyzed using the ARTIC bioinformatic pipeline for COVID-19 (https://artic.network/ncov-2019) (link should omit spaces). Consensus sequences were uploaded to GISAID (https://www.gisaid.org/) (link should omit spaces), IDs: EPI_ISL_1081321, EPI-ISL_1081322, EPI_ISL_1081323 (Elbe and Buckland-Merrett, 2017). These three SARS-CoV-2 genome sequences were identified as members of the B.1.1.7 lineage by an automated lineage assigner (Rambaut et al., 2020) (https://github.com/hCoV-2019/pangolin) (link should omit spaces).
[0115] Statistical Analyses
[0116] All experiments were performed in triplicate or duplicate and error is reported as ±1 standard deviation. The merged datasets of replicates of fluorescence kinetics of direct Csm-based detection of SARS-CoV-2 RNA in patient samples was fit to a simple linear regression, in Prism 9 (Graphpad). The fitted slopes of SARS-CoV-2 RNA-containing patient samples were compared pairwise to the negative swab RNA control by an F-test, ****p<0.0001.
Sequences
[0117]
TABLE-US-00003 TABLE 3 Examples of sequences. SEQ ID NOS. 29-138 belong to primer pool nCoV-2019_1 and SEQ ID NOS. 139-246 belong to primer pool nCoV-2019_2. SEQ ID NO. Sequence Description 1 AUUGCGACACGCUGAAGCGCUGGG RNA sequence for example GGCAAAUUGUGCAAUUUGCGGCCA CRISPR guide. RNA sequences are GUUGCAAGGGAUUGAGCCCCGUAA shown 5′ to 3′. Bases in italics GGGG correspond to portions of the CRISPR repeat. Bases that are underlined correspond to the CRISPR spacer-that is complementary to the SARS-CoV-2 N gene. 2 AUUGCGACACGCUGAAGCGCUGGG RNA sequence for example GGCAAAUUGUGCAAUUUGCGGCCA CRISPR guide. RNA sequences are GUUGCAAG shown 5′ to 3′. Bases in italics correspond to portions of the CRISPR repeat. Bases that are underlined correspond to the CRISPR spacer-that is complementary to the SARS-CoV-2 N gene. 3 AUUGCGACACGCUGAAGCGCUGGG N1 (76 nt guide) GGCAAAUUGUGCAAUUUGCGGCCA GUUGCAAGGGAUUGAGCCCCGUAA GGGG 4 AUUGCGACGGCCGACGUUGUUUUG N3 (76 nt guide) AUCGCGCCCCACUGCGUUCUCCAU GUUGCAAGGGAUUGAGCCCCGUAA GGGG 5 AUUGCGACGUUGCGACUACGUGAU N6 (76 nt guide) GAGGAACGAGAAGAGGCUUGACU GGUUGCAAGGGAUUGAGCCCCGUA AGGGG 6 AUUGCGACAGCAGCAGCAAAGCAA N7 (76 nt guide) GAGCAGCAUCACCGCCAUUGCCAG GUUGCAAGGGAUUGAGCCCCGUAA GGGG 7 AUUGCGACAUGCUUUAGUGGCAG N8 (76 nt guide) UACGUUUUUGCCGAGGCUUCUUAG AGUUGCAAGGGAUUGAGCCCCGUA AGGGG 8 AUUGCGACUUCCGAAGAACGCUGA N9 (76 nt guide) AGCGCUGGGGGCAAAUUGUGCAA UGUUGCAAGGGAUUGAGCCCCGUA AGGGG 9 AUUGCGACAUUCAGCAAAAUGACU N10 (76 nt guide) UGAUCUUUGAAAUUUGGAUCUUU GGUUGCAAGGGAUUGAGCCCCGUA AGGGG 10 AUUGCGACCAGUUUGCUGUUUCUU N11 (76 nt guide) CUGUCUCUGCGGUAAGGCUUGAGU GUUGCAAGGGAUUGAGCCCCGUAA GGGG 11 AUUGCGACGUCAGCACUGCUCAUG N12 (76 nt guide) GAUUGUUGCAAUUGUUUGGAGAA AGUUGCAAGGGAUUGAGCCCCGUA AGGGG 12 AUUGCGACAAAAGCGAAAACGUU I1 (76 nt guide) UAUAUAGCCCAUCUGCCUUGUGUG GGUUGCAAGGGAUUGAGCCCCGUA AGGGG 13 GATAATACGACTCACTATAGGGAA SARS-CoV-2 N1 T7 template CTGATTACAAACATTGGCCGCAAAT Forward Primer TGCACAATT 14 GCGCGACATTCCGAAGAACGCTGA SARS-CoV-2 N1 T7 template AGCGCTGGGGGCAAATTGTGCAAT Reverse Primer TTGCGGCC 15 GATAATACGACTCACTATAGGGAA SARS-CoV-1 N1 T7 template CTGATTACAAACATTGGCCGCAAAT Forward Primer TGCACAATT 16 GCGTGACATTCCAAAGAATGCAGA SARS-CoV-1 N1 T7 template GGCACTTGGAGCAAATTGTGCAATT Reverse Primer TGCGGCC 17 CTAGAGCTCGATAATACGACTCACT SARS-CoV-2 target all ten ATAGGGCGTGTTGTTTTAGATTTCA complexes Forward Primer TCTAAACG 18 ATCCTGCAGGCACACTGATTAAAG SARS-CoV-2 target all ten ATTGCTATGTG complexes Reverse Primer 19 GCTGCTGAGGCTTCTAAG RT-LAMP Primer F3 20 GCGTCAATATGCTTATTCAGC RT-LAMP Primer B3 21 TCAGCGTTCTTCGGAATGTCGCTGT RT-LAMP Primer BIP GTAGGTCAACCACG 22 GCGGCCAATGTTTGTAATCAGTAGA RT-LAMP Primer FIP CGTGGTCCAGAACAA 23 CCTTGTCTGATTAGTTCCTGGT RT-LAMP Primer Loop Forward 24 TGGCATGGAAGTCACACC RT-LAMP Primer Loop Reverse 25 TAATACGACTCACTATAGGGAGAC RT-LAMP Primer T7-FIP GTGGTCCAGAACAA 26 MKLKKVIRIRSVLLAKTGLRIGMSRD Amino Acid sequence of Wildtype QMAIGDLDNPVVRNPLTDEPYIPGSS TtCsm3 sequence LKGKLRYLLEWSLGGDYILKAKERQ VYASPDPKDPVARIFGLAPENDERSL AVARERGPTRLLVRDAYLTEDAKEA LERTSARGGLYTEIKQEVFIPRLGGN ANPRTTERVPAGARFRVEMTYRVLD DLDEEYFGKYLLRALELLELDGLGG HISRGYGQVYFLHPERLTEDQEGWPL KERLKVEEVVL 27 MKLKKVIRIRSVLLAKTGLRIGMSRDQMAIG Amino Acid sequence of Mutant DLANPVVRNPLTDEPYIPGSSLKGKLRYLLE TtCsm3 sequence (showing amino WSLGGDYILKAKERQVYASPDPKDPVARIFG acid mutation in bold, underline) LAPENDERSLAVARERGPTRLLVRDAYLTED AKEALERTSARGGLYTEIKQEVFIPRLGGNA NPRTTERVPAGARFRVEMTYRVLDDLDEEY FGKYLLRALELLELDGLGGHISRGYGQVYFL HPERLTEDQEGWPLKERLKVEEVVL 28 TAATACGACTCACTATAGGGagacgtg The T7 promoter sequence is in gtccagaacaa CAPS in this primer. The lowerCase bases are the same as those found in the 3′ end of the Forward Inner Primer. 29 ACCAACCAACTTTCGATCTCTTGT nCoV-2019_1_LEFT 30 CATCTTTAAGATGTTGACGTGCCTC nCoV-2019_1_RIGHT 31 CGGTAATAAAGGAGCTGGTGGC nCoV-2019_3_LEFT 32 AAGGTGTCTGCAATTCATAGCTCT nCoV-2019_3_RIGHT 33 TGGTGAAACTTCATGGCAGACG nCoV-2019_5_LEFT 34 ATTGATGTTGACTTTCTCTTTTTGGA nCoV-2019_5_RIGHT GT 35 ATCAGAGGCTGCTCGTGTTGTA nCoV-2019_7_LEFT 36 CATTTGCATCAGAGGCTGCTCG nCoV-2019_7_LEFT_alt0 37 TGCACAGGTGACAATTTGTCCA nCoV-2019_7_RIGHT 38 AGGTGACAATTTGTCCACCGAC nCoV-2019_7_RIGHT_alt5 39 TCCCACAGAAGTGTTAACAGAGGA nCoV-2019_9_LEFT 40 TTCCCACAGAAGTGTTAACAGAGG nCoV-2019_9_LEFT_alt4 41 ATGACAGCATCTGCCACAACAC nCoV-2019_9_RIGHT 42 GACAGCATCTGCCACAACACAG nCoV-2019_9_RIGHT_alt2 43 GGAATTTGGTGCCACTTCTGCT nCoV-2019_11_LEFT 44 TCATCAGATTCAACTTGCATGGCA nCoV-2019_11_RIGHT 45 TCGCACAAATGTCTACTTAGCTGT nCoV-2019_13_LEFT 46 ACCACAGCAGTTAAAACACCCT nCoV-2019_13_RIGHT 47 ACAGTGCTTAAAAAGTGTAAAAGT nCoV-2019_15_LEFT GCC 48 AGTGCTTAAAAAGTGTAAAAGTGC nCoV-2019_15_LEFT_alt1 CT 49 AACAGAAACTGTAGCTGGCACT nCoV-2019_15_RIGHT 50 ACTGTAGCTGGCACTTTGAGAGA nCoV-2019_15_RIGHT_alt3 51 CTTCTTTCTTTGAGAGAAGTGAGGA nCoV-2019_17_LEFT CT 52 TTTGTTGGAGTGTTAACAATGCAGT nCoV-2019_17_RIGHT 53 GCTGTTATGTACATGGGCACACT nCoV-2019_19_LEFT 54 TGTCCAACTTAGGGTCAATTTCTGT nCoV-2019_19_RIGHT 55 TGGCTATTGATTATAAACACTACAC nCoV-2019_21_LEFT ACCC 56 GGCTATTGATTATAAACACTACACA nCoV-2019_21_LEFT_alt2 CCCT 57 TAGATCTGTGTGGCCAACCTCT nCoV-2019_21_RIGHT 58 GATCTGTGTGGCCAACCTCTTC nCoV-2019_21_RIGHT_alt0 59 ACAACTACTAACATAGTTACACGGT nCoV-2019_23_LEFT GT 60 ACCAGTACAGTAGGTTGCAATAGT nCoV-2019_23_RIGHT G 61 GCAATTGTTTTTCAGCTATTTTGCA nCoV-2019_25_LEFT GT 62 ACTGTAGTGACAAGTCTCTCGCA nCoV-2019_25_RIGHT 63 ACTACAGTCAGCTTATGTGTCAACC nCoV-2019_27_LEFT 64 AATACAAGCACCAAGGTCACGG nCoV-2019_27_RIGHT 65 ACTTGTGTTCCTTTTTGTTGCTGC nCoV-2019_29_LEFT 66 AGTGTACTCTATAAGTTTTGATGGT nCoV-2019_29_RIGHT GTGT 67 TTCTGAGTACTGTAGGCACGGC nCoV-2019_31_LEFT 68 ACAGAATAAACACCAGGTAAGAAT nCoV-2019_31_RIGHT GAGT 69 ACTTTTGAAGAAGCTGCGCTGT nCoV-2019_33_LEFT 70 TGGACAGTAAACTACGTCATCAAG nCoV-2019_33_RIGHT C 71 TGTTCGCATTCAACCAGGACAG nCoV-2019_35_LEFT 72 ACTTCATAGCCACAAGGTTAAAGTC nCoV-2019_35_RIGHT A 73 ACACACCACTGGTTGTTACTCAC nCoV-2019_37_LEFT 74 GTCCACACTCTCCTAGCACCAT nCoV-2019_37_RIGHT 75 AGTATTGCCCTATTTTCTTCATAAC nCoV-2019_39_LEFT TGGT 76 TGTAACTGGACACATTGAGCCC nCoV-2019_39_RIGHT 77 GTTCCCTTCCATCATATGCAGCT nCoV-2019_41_LEFT 78 TGGTATGACAACCATTAGTTTGGCT nCoV-2019_41_RIGHT 79 TACGACAGATGTCTTGTGCTGC nCoV-2019_43_LEFT 80 AGCAGCATCTACAGCAAAAGCA nCoV-2019_43_RIGHT 81 TACCTACAACTTGTGCTAATGACCC nCoV-2019_45_LEFT 82 AGTATGTACAAATACCTACAACTTG nCoV-2019_45_LEFT_alt2 TGCT 83 AAATTGTTTCTTCATGTTGGTAGTT nCoV-2019_45_RIGHT AGAGA 84 TTCATGTTGGTAGTTAGAGAAAGTG nCoV-2019_45_RIGHT_alT7 TGTC 85 AGGACTGGTATGATTTTGTAGAAA nCoV-2019_47_LEFT ACCC 86 AATAACGGTCAAAGAGTTTTAACCT nCoV-2019_47_RIGHT CTC 87 AGGAATTACTTGTGTATGCTGCTGA nCoV-2019_49_LEFT 88 TGACGATGACTTGGTTAGCATTAAT nCoV-2019_49_RIGHT ACA 89 TCAATAGCCGCCACTAGAGGAG nCoV-2019_51_LEFT 90 AGTGCATTAACATTGGCCGTGA nCoV-2019_51_RIGHT 91 AGCAAAATGTTGGACTGAGACTGA nCoV-2019_53_LEFT 92 AGCCTCATAAAACTCAGGTTCCC nCoV-2019_53_RIGHT 93 ACTCAACTTTACTTAGGAGGTATGA nCoV-2019_55_LEFT GCT 94 GGTGTACTCTCCTATTTGTACTTTA nCoV-2019_55_RIGHT CTGT 95 ATTCTACACTCCAGGGACCACC nCoV-2019_57_LEFT 96 GTAATTGAGCAGGGTCGCCAAT nCoV-2019_57_RIGHT 97 TCACGCATGATGTTTCATCTGCA nCoV-2019_59_LEFT 98 AAGAGTCCTGTTACATTTTCAGCTT nCoV-2019_59_RIGHT G 99 TGTTTATCACCCGCGAAGAAGC nCoV-2019_61_LEFT 100 ATCACATAGACAACAGGTGCGC nCoV-2019_61_RIGHT 101 TGTTAAGCGTGTTGACTGGACT nCoV-2019_63_LEFT 102 ACAAACTGCCACCATCACAACC nCoV-2019_63_RIGHT 103 GCTGGCTTTAGCTTGTGGGTTT nCoV-2019_65_LEFT 104 TGTCAGTCATAGAACAAACACCAA nCoV-2019_65_RIGHT TAGT 105 GTTGTCCAACAATTACCTGAAACTT nCoV-2019_67_LEFT ACT 106 CAACCTTAGAAACTACAGATAAAT nCoV-2019_67_RIGHT CTTGGG 107 TGTCGCAAAATATACTCAACTGTGT nCoV-2019_69_LEFT CA 108 TCTTTATAGCCACGGAACCTCCA nCoV-2019_69_RIGHT 109 ACAAATCCAATTCAGTTGTCTTCCT nCoV-2019_71_LEFT ATTC 110 TGGAAAAGAAAGGTAAGAACAAGT nCoV-2019_71_RIGHT CCT 111 CAATTTTGTAATGATCCATTTTTGG nCoV-2019_73_LEFT GTGT 112 CACCAGCTGTCCAACCTGAAGA nCoV-2019_73_RIGHT 113 AGAGTCCAACCAACAGAATCTATT nCoV-2019_75_LEFT GT 114 ACCACCAACCTTAGAATCAAGATT nCoV-2019_75_RIGHT GT 115 CCAGCAACTGTTTGTGGACCTA nCoV-2019_77_LEFT 116 CAGCCCCTATTAAACAGCCTGC nCoV-2019_77_RIGHT 117 GTGGTGATTCAACTGAATGCAGC nCoV-2019_79_LEFT 118 CATTTCATCTGTGAGCAAAGGTGG nCoV-2019_79_RIGHT 119 GCACTTGGAAAACTTCAAGATGTG nCoV-2019_81_LEFT G 120 GTGAAGTTCTTTTCTTGTGCAGGG nCoV-2019_81_RIGHT 121 TCCTTTGCAACCTGAATTAGACTCA nCoV-2019_83_LEFT 122 TTTGACTCCTTTGAGCACTGGC nCoV-2019_83_RIGHT 123 ACTAGCACTCTCCAAGGGTGTT nCoV-2019_85_LEFT 124 ACACAGTCTTTTACTCCAGATTCCC nCoV-2019_85_RIGHT 125 CGACTACTAGCGTGCCTTTGTA nCoV-2019_87_LEFT 126 ACTAGGTTCCATTGTTCAAGGAGC nCoV-2019_87_RIGHT 127 GTACGCGTTCCATGTGGTCATT nCoV-2019_89_LEFT 128 CGCGTTCCATGTGGTCATTCAA nCoV-2019_89_LEFT_alt2 129 ACCTGAAAGTCAACGAGATGAAAC nCoV-2019_89_RIGHT A 130 ACGAGATGAAACATCTGTTGTCACT nCoV-2019_89_RIGHT_alt4 131 TCACTACCAAGAGTGTGTTAGAGGT nCoV-2019_91_LEFT 132 TTCAAGTGAGAACCAAAAGATAAT nCoV-2019_91_RIGHT AAGCA 133 TGAGGCTGGTTCTAAATCACCCA nCoV-2019_93_LEFT 134 AGGTCTTCCTTGCCATGTTGAG nCoV-2019_93_RIGHT 135 TGAGGGAGCCTTGAATACACCA nCoV-2019_95_LEFT 136 CAGTACGTTTTTGCCGAGGCTT nCoV-2019_95_RIGHT 137 TGGATGACAAAGATCCAAATTTCA nCoV-2019_97_LEFT AAGA 138 ACACACTGATTAAAGATTGCTATGT nCoV-2019_97_RIGHT GAG 139 CTGTTTTACAGGTTCGCGACGT nCoV-2019_2_LEFT 140 TAAGGATCAGTGCCAAGCTCGT nCoV-2019_2_RIGHT 141 GGTGTATACTGCTGCCGTGAAC nCoV-2019_4_LEFT 142 CACAAGTAGTGGCACCTTCTTTAGT nCoV-2019_4_RIGHT 143 GGTGTTGTTGGAGAAGGTTCCG nCoV-2019_6_LEFT 144 TAGCGGCCTTCTGTAAAACACG nCoV-2019_6_RIGHT 145 AGAGTTTCTTAGAGACGGTTGGGA nCoV-2019_8_LEFT 146 GCTTCAACAGCTTCACTAGTAGGT nCoV-2019_8_RIGHT 147 TGAGAAGTGCTCTGCCTATACAGT nCoV-2019_10_LEFT 148 TCATCTAACCAATCTTCTTCTTGCTC nCoV-2019_10_RIGHT T 149 AAACATGGAGGAGGTGTTGCAG nCoV-2019_12_LEFT 150 TTCACTCTTCATTTCCAAAAAGCTT nCoV-2019_12_RIGHT GA 151 CATCCAGATTCTGCCACTCTTGT nCoV-2019_14_LEFT 152 TGGCAATCTTCATCCAGATTCTGC nCoV-2019_14_LEFT_alt4 153 AGTTTCCACACAGACAGGCATT nCoV-2019_14_RIGHT 154 TGCGTGTTTCTTCTGCATGTGC nCoV-2019_14_RIGHT_alt2 155 AATTTGGAAGAAGCTGCTCGGT nCoV-2019_16_LEFT 156 CACAACTTGCGTGTGGAGGTTA nCoV-2019_16_RIGHT 157 TGGAAATACCCACAAGTTAATGGTT nCoV-2019_18_LEFT TAAC 158 ACTTCTATTAAATGGGCAGATAACA nCoV-2019_18_LEFT_alt2 ACTGT 159 AGCTTGTTTACCACACGTACAAGG nCoV-2019_18_RIGHT 160 GCTTGTTTACCACACGTACAAGG nCoV-2019_18_RIGHT_alt1 161 ACAAAGAAAACAGTTACACAACAA nCoV-2019_20_LEFT CCA 162 ACGTGGCTTTATTAGTTGCATTGTT nCoV-2019_20_RIGHT 163 ACTACCGAAGTTGTAGGAGACATT nCoV-2019_22_LEFT ATACT 164 ACAGTATTCTTTGCTATAGTAGTCG nCoV-2019_22_RIGHT GC 165 AGGCATGCCTTCTTACTGTACTG nCoV-2019_24_LEFT 166 ACATTCTAACCATAGCTGAAATCGG nCoV-2019_24_RIGHT G 167 TTGTGATACATTCTGTGCTGGTAGT nCoV-2019_26_LEFT 168 TCCGCACTATCACCAACATCAG nCoV-2019_26_RIGHT 169 ACATAGAAGTTACTGGCGATAGTT nCoV-2019_28_LEFT GT 170 TGTTTAGACATGACATGAACAGGT nCoV-2019_28_RIGHT GT 171 GCACAACTAATGGTGACTTTTTGCA nCoV-2019_30_LEFT 172 ACCACTAGTAGATACACAAACACC nCoV-2019_30_RIGHT AG 173 TGGTGAATACAGTCATGTAGTTGCC nCoV-2019_32_LEFT 174 AGCACATCACTACGCAACTTTAGA nCoV-2019_32_RIGHT 175 TCCCATCTGGTAAAGTTGAGGGT nCoV-2019_34_LEFT 176 AGTGAAATTGGGCCTCATAGCA nCoV-2019_34_RIGHT 177 TTAGCTTGGTTGTACGCTGCTG nCoV-2019_36_LEFT 178 GAACAAAGACCATTGAGTACTCTG nCoV-2019_36_RIGHT GA 179 ACTGTGTTATGTATGCATCAGCTGT nCoV-2019_38_LEFT 180 CACCAAGAGTCAGTCTAAAGTAGC nCoV-2019_38_RIGHT G 181 TGCACATCAGTAGTCTTACTCTCAG nCoV-2019_40_LEFT T 182 CATGGCTGCATCACGGTCAAAT nCoV-2019_40_RIGHT 183 TGCAAGAGATGGTTGTGTTCCC nCoV-2019_42_LEFT 184 CCTACCTCCCTTTGTTGTGTTGT nCoV-2019_42_RIGHT 185 TGCCACAGTACGTCTACAAGCT nCoV-2019_44_LEFT 186 CCACAGTACGTCTACAAGCTGG nCoV-2019_44_LEFT_alt3 187 AACCTTTCCACATACCGCAGAC nCoV-2019_44_RIGHT 188 CGCAGACGGTACAGACTGTGTT nCoV-2019_44_RIGHT_alt0 189 TGTCGCTTCCAAGAAAAGGACG nCoV-2019_46_LEFT 190 CGCTTCCAAGAAAAGGACGAAGA nCoV-2019_46_LEFT_alt1 191 CACGTTCACCTAAGTTGGCGTA nCoV-2019_46_RIGHT 192 CACGTTCACCTAAGTTGGCGTAT nCoV-2019_46_RIGHT_alt2 193 TGTTGACACTGACTTAACAAAGCCT nCoV-2019_48_LEFT 194 TAGATTACCAGAAGCAGCGTGC nCoV-2019_48_RIGHT 195 GTTGATAAGTACTTTGATTGTTACG nCoV-2019_50_LEFT ATGGT 196 TAACATGTTGTGCCAACCACCA nCoV-2019_50_RIGHT 197 CATCAGGAGATGCCACAACTGC nCoV-2019_52_LEFT 198 GTTGAGAGCAAAATTCATGAGGTC nCoV-2019_52_RIGHT C 199 TGAGTTAACAGGACACATGTTAGA nCoV-2019_54_LEFT CA 200 AACCAAAAACTTGTCCATTAGCAC nCoV-2019_54_RIGHT A 201 ACCTAGACCACCACTTAACCGA nCoV-2019_56_LEFT 202 ACACTATGCGAGCAGAAGGGTA nCoV-2019_56_RIGHT 203 TGATTTGAGTGTTGTCAATGCCAGA nCoV-2019_58_LEFT 204 CTTTTCTCCAAGCAGGGTTACGT nCoV-2019_58_RIGHT 205 TGATAGAGACCTTTATGACAAGTTG nCoV-2019_60_LEFT CA 206 GGTACCAACAGCTTCTCTAGTAGC nCoV-2019_60_RIGHT 207 GGCACATGGCTTTGAGTTGACA nCoV-2019_62_LEFT 208 GTTGAACCTTTCTACAAGCCGC nCoV-2019_62_RIGHT 209 TCGATAGATATCCTGCTAATTCCAT nCoV-2019_64_LEFT TGT 210 AGTCTTGTAAAAGTGTTCCAGAGGT nCoV-2019_64_RIGHT 211 GGGTGTGGACATTGCTGCTAAT nCoV-2019_66_LEFT 212 TCAATTTCCATTTGACTCCTGGGT nCoV-2019_66_RIGHT 213 ACAGGTTCATCTAAGTGTGTGTGT nCoV-2019_68_LEFT 214 CTCCTTTATCAGAACCAGCACCA nCoV-2019_68_RIGHT 215 ACAAAAGAAAATGACTCTAAAGAG nCoV-2019_70_LEFT GGTTT 216 TGACCTTCTTTTAAAGACATAACAG nCoV-2019_70_RIGHT CAG 217 ACACGTGGTGTTTATTACCCTGAC nCoV-2019_72_LEFT 218 ACTCTGAACTCACTTTCCATCCAAC nCoV-2019_72_RIGHT 219 ACATCACTAGGTTTCAAACTTTACT nCoV-2019_74_LEFT TGC 220 GCAACACAGTTGCTGATTCTCTTC nCoV-2019_74_RIGHT 221 AGGGCAAACTGGAAAGATTGCT nCoV-2019_76_LEFT 222 GGGCAAACTGGAAAGATTGCTGA nCoV-2019_76_LEFT_alt3 223 ACACCTGTGCCTGTTAAACCAT nCoV-2019_76_RIGHT 224 ACCTGTGCCTGTTAAACCATTGA nCoV-2019_76_RIGHT_alt0 225 CAACTTACTCCTACTTGGCGTGT nCoV-2019_78_LEFT 226 TGTGTACAAAAACTGCCATATTGCA nCoV-2019_78_RIGHT 227 TTGCCTTGGTGATATTGCTGCT nCoV-2019_80_LEFT 228 TGGAGCTAAGTTGTTTAACAAGCG nCoV-2019_80_RIGHT 229 GGGCTATCATCTTATGTCCTTCCCT nCoV-2019_82_LEFT 230 TGCCAGAGATGTCACCTAAATCAA nCoV-2019_82_RIGHT 231 TGCTGTAGTTGTCTCAAGGGCT nCoV-2019_84_LEFT 232 AGGTGTGAGTAAACTGTTACAAAC nCoV-2019_84_RIGHT AAC 233 TCAGGTGATGGCACAACAAGTC nCoV-2019_86_LEFT 234 ACGAAAGCAAGAAAAAGAAGTACG nCoV-2019_86_RIGHT C 235 CCATGGCAGATTCCAACGGTAC nCoV-2019_88_LEFT 236 TGGTCAGAATAGTGCCATGGAGT nCoV-2019_88_RIGHT 237 ACACAGACCATTCCAGTAGCAGT nCoV-2019_90_LEFT 238 TGAAATGGTGAATTGCCCTCGT nCoV-2019_90_RIGHT 239 TTTGTGCTTTTTAGCCTTTCTGCT nCoV-2019_92_LEFT 240 AGGTTCCTGGCAATTAATTGTAAAA nCoV-2019_92_RIGHT GG 241 GGCCCCAAGGTTTACCCAATAA nCoV-2019_94_LEFT 242 TTTGGCAATGTTGTTCCTTGAGG nCoV-2019_94_RIGHT 243 GCCAACAACAACAAGGCCAAAC nCoV-2019_96_LEFT 244 TAGGCTCTGTTGGTGGGAATGT nCoV-2019_96_RIGHT 245 AACAATTGCAACAATCCATGAGCA nCoV-2019_98_LEFT 246 TTCTCCTAAGAAGCTATTAAAATCA nCoV-2019_98_RIGHT CATGG
[0118] A subject may refer to an animal, such as a mammalian species (preferably human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals, sport animals, and pets. A subject can be a healthy individual, an individual that has symptoms or signs or is suspected of having a disease or a predisposition to the disease, or an individual that is in need of therapy or suspected of needing therapy.
[0119] A genetic modification or mutation in the context of an engineered system may refer to an alteration, variant or polymorphism in a nucleic acid that may result in altered or disabled functionality of a corresponding protein. Such alteration, variant or polymorphism can be with respect to a reference genome, the subject or other individual. Variations include one or more single nucleotide variations (SNVs), insertions, deletions, repeats, small insertions, small deletions, small repeats, structural variant junctions, variable length tandem repeats, and/or flanking sequences, CNVs, transversions, gene fusions and other rearrangements may also be considered forms of genetic variation. A variation can be a base change, insertion, deletion, repeat, copy number variation, transversion, or a combination thereof.
[0120] A “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, or “oligonucleotide” may each refer to a polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g. 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5′.fwdarw.3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted. The letters A, C, G, and T (or “U” denoting Uracil in RNA) may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.
[0121] A “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, or “oligonucleotide” may each refer to a polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g. 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5′.fwdarw.3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted. The letters A, C, G, and T (or “U” denoting Uracil in RNA) may be used to refer to the bases themselves, to nucleosides, or
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[0162] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.