Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Specific commercial products and services available to the researchers to implement CRISPR technology.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Genome Wide, Clone Assay, Stable Transfection, Selection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Negative Control, Positive Control, Construct, Knock-In, Multiplex Assay, Amplification, Sequencing

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: (A) NP swab diluents from 2 confirmed COVID-19 patients were pooled, and using the 2019-nCoV_N3 primer/probe set, the mixture was either (i) subjected to RNA extraction using the Qiagen QIAamp Viral RNA Mini Kit followed by subsequent testing by RT-qPCR (using the equivalent of 11.3 μl of swab diluent) or (ii) directly added to the RT-qPCR reaction, with or without a preheating step (5 minutes at 70°C, “NP sample + heat”). As a control, the indicated quantities of the CDC 2019-nCoV Positive Control SARS-CoV-2 synthetic RNA were spiked into M6 transport medium, purified using the QIAamp Viral RNA Mini Kit, and screened by RT-qPCR. NP swab samples from 7 additional donors were screened by direct RT-qPCR for SARS-CoV-2 RNA using the 2019-nCoV_N1 primer/probe set (B) or the 2019-nCoV_N2 primer/probe set (C), or for human RNase P RNA using the RNase P primer/probe set (D). NP swab samples from donors 1–4 were previously shown to contain SARS-CoV-2 RNA by standard clinical RT-qPCR, while donors 5–7 were negative. For each primer/probe set, 7 μl (A) or 3 μl (B–D) of NP swab diluent was tested in the RT-qPCR reaction per donor. For the N1 and N2 primer/probe sets, the fully synthetic SARS-CoV-2 RNA Control 2 from Twist Bioscience was loaded at serial 10-fold dilutions (A, 3 × 10 6 copies; B, 3 × 10 5 copies; C, 3 × 10 4 copies; D, 3 × 10 3 copies; E, 3 × 10 2 copies; F, 3 × 10 1 copies) as indicated in (B) and (C). NTC wells were included for each primer/probe set, and each was negative. For (B) and (C), the correlation coefficients ( R 2 ) of the standard curves were 0.999 and 0.995, respectively. The dashed line at cycle 40 in each graph indicates the limit of detection. CDC, Centers for Disease Control and Prevention; CT, cycle threshold; NP, nasopharyngeal; NTC, no template control; RT-qPCR, reverse transcription–quantitative polymerase chain reaction.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: RNA Extraction, Quantitative RT-PCR, Positive Control, Purification, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: Detection of SARS-CoV-2 RNA from NP swab diluent by direct RT-qPCR and the impact of heat and loading volume on assay sensitivity.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques:

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: Detection sensitivity of direct RT-qPCR versus standard RT-qPCR on NP swabs containing a range of SARS-CoV-2 viral RNA loads.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques:

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: A total of 150 NP swab samples representing high (CT values less than 20), intermediate (CT values of 20–30), or low (CT values of more than 30) SARS-CoV-2 RNA loads as determined by standard clinical RT-qPCR at the University of Washington in Seattle (aqua circles) were analyzed by the indicated method. All assays used the 2019-nCoV_N2 primer/probe set. Direct RT-qPCR was performed on 3 μl of NP swab diluent after heating for 10 minutes at 95°C (green circles). In parallel, RNA was extracted from 30 μl of NP swab diluent that had been previously heated at 95°C for 10 minutes, and RNA representing 3 μl of the original diluent was used in RT-qPCR (purple circles) to allow a head-to-head comparison with direct RT-qPCR on the same quantity of NP swab diluent. The limit of detection (CT of 40) is denoted with a dashed line. Samples with CT values above this cutoff were considered negative for SARS-CoV-2 RNA. The fitted curves are LOESS (locally estimated scatterplot smoothing)–smoothed CT values, with 95% confidence intervals in gray, against the mean of CT values detected in the clinical RT-qPCR assay with primer sets N1 and N2. Samples are ordered by the latter mean. The full dataset for this experiment and controls are provided in . CT, cycle threshold; NP, nasopharyngeal; RT-qPCR, reverse transcription–quantitative polymerase chain reaction.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: A total of 60 NP swab samples representing low loads (CT of 27–36) of SARS-CoV-2 RNA as determined by standard clinical RT-qPCR at UW in Seattle (purple circles) were analyzed by the indicated method. All assays used the 2019-nCoV_N2 primer/probe set. Direct RT-qPCR was performed on 3 μl of NP swab diluent after heating for 10 minutes at 95°C (green circles). In parallel, RNA was newly extracted from 200 μl of NP swab diluent (aqua circles) and processed with the UW LDT to control for the effect of freeze/thaw cycles. The limit of detection (CT of 40) is denoted with a red dashed line. Samples with CT values above this cutoff were considered negative for SARS-CoV-2 RNA. The fitted curves are LOESS (locally estimated scatterplot smoothing)–smoothed CT values, with 95% confidence intervals in gray, against the CT values detected in the 200-μl freshly extracted RT-qPCR assay with primer set N2. Samples are ordered by the CT value of freshly extracted 200-μl LDT samples. The full dataset for this experiment and controls are provided in . CT, cycle threshold; LDT, laboratory developed test; NP, nasopharyngeal; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; UW, University of Washington.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: RT-qPCR conditions.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques:

Journal: bioRxiv

Article Title: Base-edited CAR T Cells for combinational therapy against T cell malignancies

doi: 10.1101/2020.07.30.228429

Figure Lengend Snippet: A) Schema of base editing for T cells employing 3 rd generation codon optimised cytidine base deaminase (coBE3) fused to deactivated D10A Cas9 nickase and uracil glycosylase inhibitor (UGI) delivered as mRNA along with TRBC and CD7 single guide RNA (sgRNA). C->U->T conversion (G->A antisense strand) resulting in STOP codon. B) Lentiviral transduction of edited cells from step 1 using 3 rd generation lentiviral vectors. Lentiviral plasmid configuration of CD3ε targeting 2 nd generation chimeric antigen receptor comprising OKT3 vL and vH scFv sequence fused to CD8 transmembrane domain (TM), 41BB co-stimulatory and CD3z activation domains under the control of a hPGK promoter. Lentiviral plasmid configuration of CD7 targeting 2 nd generation CAR comprising 3A1e vL and vH scFv sequence fused to CD8TM-41BB-CD3z under the control of a hPGK. C) coBE3 edited T cells devoid of shared antigens TCR/CD3 and CD7 surface receptors expressing either 3CAR or 7CAR evade fratricide and target T-ALL. BE: base editor; APOBEC: (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like); sgRNA: single guide RNA; PAM: protospacer adjacent motif; LTR: long terminal repeat; CMV: cytomegalovirus promoter; CAR: chimeric antigen receptor; cPPT: central polypurine track; U5: untranslated 5’ region; DU3: delta untranslated 3’ region; hPGK: human phosphoglycerate kinase promoter; vL: variable light chain; vH: variable heavy chain.

Article Snippet: HiFi Cas9 Nuclease V3 (1081061, IDT, Leuven, Belgium) was used as positive control.

Techniques: Transduction, Plasmid Preparation, Sequencing, Activation Assay, Control, Expressing

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Schematic representation of pTRM4-TAA expression plasmid. The wild-type TRM4 gene is mutated at F602 position (TTT converted to TAA) to generate a premature termination codon (PTC) (indicated). The plasmid-borne TRM4 gene is fused with a multicomponent C-terminal tag to facilitate the detection and purification of full-length Trm4 product. The tag contains a 3C protease cleavage site that can be used to release the purified protein from the tag. The expression of TRM4 gene is under the control of Gal promoter, which is galactose-inducible. URA3 is an auxotroph selective marker in yeast. This is a high copy 2 µ plasmid.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Expressing, Plasmid Preparation, Purification, Marker

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: The strategy for cloning artificial snR81 using four primers overlapping PCR (also see Fig. 2). The construction is based on naturally occurring yeast snR81 box H/ACA RNA. The 5′-most primer is sense-strand sequence (forward), while the other primers have antisense sequences (reverse). The guide sequences (guide1, guide1′, guide2, and guide2′) are changed to base pair with substrate mRNAs, while the rest of the snR81 sequence remain unchanged. The amplified artificial snR81 is digested with BamHI and HindIII and subsequently cloned into pSEC plasmid under the control of the Gal promoter. LEU2 is an auxotroph selective marker in yeast.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Clone Assay, Sequencing, Amplification, Plasmid Preparation, Marker

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Schematic representation of site-specific RNase H cleavage of TRM4 mRNA directed by 2′-O-methyl RNA–DNA chimera. The target sequence of TRM4 mRNA is shown and the PTC (UAA) is indicated. The antisense TRM4 2′-O-methyl RNA–DNA chimera is also shown. d represents deoxy, and m stands for 2′-O-methyl. The arrow indicates the RNase H cleavage site.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Sequencing

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: TLC analysis of uridine-to-Ψ conversion at the PTC within the TRM4 mRNA transcript. The PTC-containing TRM4 mRNA, coexpressed with a PTC-specific guide RNA, is purified by oligonucleotide affinity chromatography. The RNA is cleaved by RNase H (directed by a specific 2′-O-methyl RNA–DNA chimera) at the site 5′ of the PTC (UAA). The resulting 3′-half fragment is 5′ radiolabeled with 32P through dephosphorylation and rephosphorylation (see text). The labeled RNA is digested with nuclease P1 to completion. The digested sample is mixed with an equal amount of 5′-32P-adenosine-monophosphate, 5′–32P-cytidine-monophosphate, and 5′–32P-guanosine-monophosphate, and analyzed by two-dimensional TLC. The first and second dimensions are indicated. The origin and the positions of each 5′-phosphorylated nucleotide are also indicated.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Purification, Affinity Chromatography, De-Phosphorylation Assay, Labeling

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Ψ-Mediated nonsense suppression detected by Western blotting. Equal amounts of total protein are loaded (lanes 1–4), and anti-Protein A and anti-GAPDH (loading control) are used for blotting. Readthrough of PTC is visualized (lane 4), where a cognate (PTC-specific) guide RNA designed to target the PTC for pseudouridylation is coexpressed. In contrast, no significant suppression is observed when no guide RNA (lane 1) or a guide RNA-containing random guide sequences is coexpressed (lane 3). Lane 2 is a positive control where the wild-type TRM4 mRNA (with no PTC) is expressed.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Western Blot, Positive Control

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation

Journal: Cell reports

Article Title: Intestinal IL-17R Signaling Constrains IL-18-Driven Liver Inflammation by the Regulation of Microbiome-Derived Products

doi: 10.1016/j.celrep.2019.10.042

Figure Lengend Snippet:

Article Snippet: Primer: mouse Hprt , Integrated DNA Technologies , Cat# Mm.PT.39a.22214828.

Techniques: Recombinant, Purification, Activity Assay, In Situ, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Staining, RNA Sequencing Assay, Sequencing, Software, SYBR Green Assay, Protease Inhibitor, Cell Counting