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1.
Science ; 372(6545): 941-948, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1205995

ABSTRACT

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp614→Gly) variant with single-base resolution in patient samples.


Subject(s)
CRISPR-Associated Protein 9/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , RNA, Guide/genetics , RNA, Viral/analysis , RNA/analysis , RNA/genetics , SARS-CoV-2/genetics , Base Sequence , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing , CRISPR-Cas Systems , Campylobacter jejuni , Humans , Nucleic Acid Hybridization , RNA, Bacterial/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Spike Glycoprotein, Coronavirus/genetics
2.
Talanta ; 228: 122227, 2021 Jun 01.
Article in English | MEDLINE | ID: covidwho-1100758

ABSTRACT

Nucleic acid detection and quantification have been known to be important at various fields, from genetically modified organisms and gene expression to virus detection. For DNA molecules, digital PCR has been developed as an absolute quantification method which is not dependent on external calibrators. While when it comes to RNA molecules, reverse transcription (RT) step must be taken before PCR amplification to obtain cDNA. With different kinds of reverse transcriptase (RTase) and RT reaction conditions being used in laboratory assays, the efficiency of RT process differs a lot which led variety in quantification results of RNA molecules. In this study, we developed HPLC method combined with enzymatic digestion of RNA to nucleotides for quantification of RNA without RT process. This method was metrologically traceable to four nuceloside monophosphate (NMP) Certification Reference Materials of National Institute of Metrology, China (NIMC) for insurance of accuracy. The established method was used to evaluate the reverse transcription digital polymerase chain reaction (RT-dPCR) of three target genes of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RNA, including open reading frame 1ab (ORF1ab), nucleocapsid protein (N) and envelope protein (E) gene. Three available RT kits had been evaluated and disparities were observed for the RT efficiency varied from 9% to 182%. It is thus demonstrated that HPLC combined with enzymatic digestion could be a useful method to quantify RNA molecules and evaluate RT efficiency. It is suggested that RT process should be optimized and identified in RNA quantification assays.


Subject(s)
Chromatography, High Pressure Liquid/methods , Phosphodiesterase I/chemistry , Proteolysis , RNA/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Chromatography, High Pressure Liquid/standards , Coronavirus Nucleocapsid Proteins/genetics , Crotalinae , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/genetics , Purine Nucleotides/standards , Pyrimidine Nucleotides/standards , RNA/chemistry , Reference Standards
3.
Anal Methods ; 13(2): 169-178, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1039652

ABSTRACT

We demonstrate a loop-mediated isothermal amplification (LAMP) method to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and RNA extracts from patients, we demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR (i.e., sensitivity of 92.85% and specificity of 81.25% in a set of 44 RNA extracts from patients analyzed in a hospital setting).


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA/analysis , SARS-CoV-2/chemistry , Viral Load/methods , COVID-19/diagnosis , Colorimetry/methods , Coronavirus Nucleocapsid Proteins , DNA/analysis , DNA/chemistry , Fluorescent Dyes/chemistry , Humans , Intercalating Agents/chemistry , Phenolsulfonphthalein/chemistry , Phosphoproteins , RNA/chemistry
4.
ACS Nano ; 15(1): 1167-1178, 2021 01 26.
Article in English | MEDLINE | ID: covidwho-1014984

ABSTRACT

Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.


Subject(s)
CRISPR-Cas Systems , Microfluidics , RNA/analysis , Cell Line, Tumor , Enterococcus faecalis , Escherichia coli , Humans , Klebsiella pneumoniae , MCF-7 Cells , MicroRNAs/analysis , Pseudomonas aeruginosa , RNA, Ribosomal, 16S/analysis , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Staphylococcus aureus
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