nebuffer2  (New England Biolabs)

Journal: Analytical Chemistry

Article Title: Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology

doi: 10.1021/acs.analchem.0c04047

Figure Lengend Snippet: Incorporating CRISPR Cas12a-mediated detection with RT-LAMP amplification improves the specificity. (A) RT-LAMP amplification curves from triplicate analyses of the target N gene and the negative controls, with the real-time fluorescence detection of intercalating SYBR Green. (B) CRISPR Cas12a-mediated detection of the RT-LAMP products. In this set of experiments, 25 μL of RT-LAMP reaction solution contained 1× NEBuffer 2.1 buffer, 1.4 mM deoxynucleotide (dNTP), 0.2 μM each of the outer primers (F3 and B3), 1.6 μM each of the inner primers (FIP and BIP), 0.8 μM each of the loop primers (LF and LB), 4 units of RNase inhibitor, 7.5 units of WarmStart RTx reverse transcriptase, 8 units of Bst 2.0 DNA polymerase, and 5 μL of 750 copies/μL viral RNA (as target) or nuclease-free water (as negative control). RT-LAMP reactions were performed at 62 °C, and the reaction products were monitored using either SYBR Green (A) or the CRISPR Cas12a system (B).

Article Snippet: The EnGen Lba Cas12a enzyme (NEB) at 1 μM concentration was preincubated with 1.25 μM gRNA in 1× NEBuffer 2.1 (NEB) to form the ribonucleoprotein (RNP) complex.

Techniques: CRISPR, Amplification, Fluorescence, SYBR Green Assay, Negative Control

Journal: The EMBO Journal

Article Title: Accessory factors determine the order of strand exchange in Xer recombination at psi

doi: 10.1093/emboj/cdf379

Figure Lengend Snippet: Fig. 6. Xer recombination at psi and psiDC in the presence of XerC[De]. ( A ) Recombination of p- psi.psi and p- psiDC.psiDC in the presence of XerD and either XerC[De] or XerC. ( B ) Time course of recombination on p- psiDC.psiDC with XerC[De] and XerD. Reactions contained 40% glycerol and were cleaved with Xho I. Bands are indicated as follows: HJ, HJ intermediates; S, substrate fragments; P, product fragments.

Article Snippet: The other lane was excised from the gel and equilibrated for 2 h with two changes in Xho I restriction buffer (New England Biolabs NEBuffer 2 + BSA).

Techniques:

Journal: medRxiv

Article Title: A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern

doi: 10.1101/2021.10.15.21265066

Figure Lengend Snippet: BrCas12b characterization and one-pot specificity sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).

Article Snippet: In short, BrCas12b, sgRNA, and dsDNA activator were combined to a final concentration of 100 nM: 125 nM: 1 nM respectively in 1x NEBuffer 2.1 (New England Biolabs) and incubated at 62°C for 30 minutes.

Techniques: In Vitro, Cleavage Assay, Multiplexing, Amplification, Fluorescence, Standard Deviation

Journal: Scientific Reports

Article Title: Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells

doi: 10.1038/srep05105

Figure Lengend Snippet: Transient transfection experiments for validating AdV shuttle plasmids pAdSh.PGK.Cas9 and pAdSh.U6.gRNA S1 . AAVS1 -specific PCR products amplified from E1 -transformed PER.C6 cells co-transfected with hCas9 (9.6 kb) and gRNA_AAVS1-T2 (4 kb), pAdSh.PGK.Cas9 (11.6 kb) and pAdSh.U6.gRNA S1 (7.3 kb), hCas9 and “empty gRNA” construct gRNA_Cloning Vector (3.9 kb), hCas9_D10A (9.6 kb) and gRNA_AAVS1-T2 or mock-transfected. Marker, Gene Ruler DNA Ladder (Fermentas). Plasmids hCas9 and hCas9_D10A express Cas9 nucleases which induce a DSB and a nick at AAVS1 , respectively. After amplicon denaturation and reannealing, the presence of mismatches derived from NHEJ-mediated repair of site-specific DSBs in cellula was probed by T7 endonuclease I (T7EI) digestions (upper panel). Negative controls were provided by amplicons not exposed to T7EI (-T7EI) as well as T7EI-treated amplicons (+T7EI) corresponding to mock-transfected cells, to cells co-transfected with hCas9 and gRNA_Cloning Vector and to cells co-transfected with hCas9_D10A and gRNA_AAVS1-T2 encoding the “nickase” mutant version of Cas9 (i.e. Cas9 D10A ) and gRNA S1 , respectively. Solid and open arrowheads indicate the positions of, respectively, undigested and T7EI-digested DNA fragments whose sizes are consistent with DSB formation at the AAVS1 target site. % KO and UN, knockout frequency and undetected, respectively.

Article Snippet: One fifth of each PCR mixture was incubated in 15-μl reactions with 1 × NEBuffer 2 and 5 U of T7EI (both from New England Biolabs).

Techniques: Transfection, Polymerase Chain Reaction, Amplification, Transformation Assay, Construct, Clone Assay, Plasmid Preparation, Marker, Derivative Assay, Non-Homologous End Joining, Mutagenesis, Knock-Out

Journal: Scientific Reports

Article Title: Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells

doi: 10.1038/srep05105

Figure Lengend Snippet: Analyses of site-specific DSB formation in U2OS cells following AdV-mediated delivery of transgenes encoding RGN and TALEN complexes. (A) Targeted mutagenesis experiments with AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 . Agarose gel electrophoresis of AAVS1 -specific PCR products amplified from chromosomal DNA of U2OS cells co-transduced with the indicated MOIs of AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 (in TU/cell). Prior to loading the PCR products were denaturated, re-annealed and treated with the mismatch-sensitive enzyme T7EI. Solid and open arrowheads point to the positions of undigested and T7EI-digested DNA molecules, respectively. Lane M, Gene Ruler DNA Ladder marker. (B) Target gene knockout frequencies in U2OS cells co-transduced with AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 as measured by densitometry of the DNA species shown in Fig. 5A. (C) Targeted mutagenesis experiments with AdV Δ2 P.TALEN-L S1 .F 50 and AdV Δ2 P.TALEN-R S1 .F 50 . Upper panel, T7EI-based assays on AAVS1 -specific amplicons corresponding to genomic DNA of mock-transduced U2OS cells (−) or of U2OS cells co-transduced with the specified MOIs of AdV Δ2 P.TALEN-L S1 .F 50 and AdV Δ2 P.TALEN-R S1 .F 50 (in TU/cell). Solid and open arrowheads indicate the positions of undigested and T7EI-digested DNA molecules, respectively. Lanes M, Gene Ruler DNA Ladder marker. Lower panel, target gene knockout frequencies in U2OS cells co-transduced with AdV Δ2 P.TALEN-L S1 .F 50 and AdV Δ2 P.TALEN-R S1 .F 50 as determined by densitometry of the DNA fragments presented in the upper panel.

Article Snippet: One fifth of each PCR mixture was incubated in 15-μl reactions with 1 × NEBuffer 2 and 5 U of T7EI (both from New England Biolabs).

Techniques: Mutagenesis, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Transduction, Marker, Gene Knockout

Journal: Scientific Reports

Article Title: Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells

doi: 10.1038/srep05105

Figure Lengend Snippet: Transduction experiments in HeLa cells for the functional validation of AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 . (A) Genotyping assays based on the detection of indels generated by NHEJ-mediated DSB repair at the chromosomal target site. The AAVS1 -specific amplicons derived from genomic DNA of HeLa cells co-transduced with AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 at the indicated MOIs (in TU/cell) were denaturated and re-annealed, treated with T7EI and resolved through agarose gel electrophoresis. As negative controls, it was used genomic DNA isolated from HeLa cells exposed to 100 TU/cell of AdV Δ2 P.Cas9.F 50 (lane 1), 100 TU/cell of AdV Δ2 U6.gRNA S1 .F 50 (lane 2) or to no vector (lane 3). Solid and open arrowheads indicate the positions of undigested and T7EI-digested DNA molecules, respectively. Lane M, Gene Ruler DNA Ladder marker. (B) Target gene knockout frequencies in HeLa cells co-transduced with AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 as measured by densitometry of the DNA species depicted in Fig. 4A.

Article Snippet: One fifth of each PCR mixture was incubated in 15-μl reactions with 1 × NEBuffer 2 and 5 U of T7EI (both from New England Biolabs).

Techniques: Transduction, Functional Assay, Generated, Non-Homologous End Joining, Derivative Assay, Agarose Gel Electrophoresis, Isolation, Plasmid Preparation, Marker, Gene Knockout

Journal: Scientific Reports

Article Title: Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells

doi: 10.1038/srep05105

Figure Lengend Snippet: Analyses of targeted mutagenesis in human myoblasts following AdV-mediated transfer of DNA coding for RGN and TALEN complexes. (A) Targeted mutagenesis experiments with AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 . Upper panel, AAVS1 -specific PCR products corresponding to genomic DNA of myoblasts co-transduced with the indicated MOIs of AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 (in TU/cell) subjected to T7EI-based assays. Lower panel, negative controls corresponding to human myoblasts that were singly transduced with AdV Δ2 P.Cas9.F 50 (lane 1), AdV Δ2 U6.gRNA S1 .F 50 (lane 2) or AdV Δ2 P.TALEN-L S1 .F 50 (lane 3) or that were mock-transduced (lane 4). (B) Target gene knockout frequencies in human myoblasts co-transduced with AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 as measured by densitometry of the DNA species depicted in Fig. 6A. (C) Targeted mutagenesis experiments with AdV Δ2 P.TALEN-L S1 .F 50 and AdV Δ2 P.TALEN-R S1 .F 50 . Upper panel, T7EI-based assays on AAVS1 -specific amplicons obtained from genomic DNA of myoblasts co-transduced with the indicated MOIs of AdV Δ2 P.TALEN-L S1 .F 50 and AdV Δ2 P.TALEN-R S1 .F 50 (in TU/cell). Solid and open arrowheads indicate the positions of undigested and T7EI-digested DNA molecules, respectively. Lanes M, Gene Ruler DNA Ladder marker. Lower panel, target gene disruption frequencies in myoblasts co-transduced with AdV Δ2 P.TALEN-L S1 .F 50 and AdV Δ2 P.TALEN-R S1 .F 50 as determined by densitometry of the DNA fragments shown in the upper panel.

Article Snippet: One fifth of each PCR mixture was incubated in 15-μl reactions with 1 × NEBuffer 2 and 5 U of T7EI (both from New England Biolabs).

Techniques: Mutagenesis, Polymerase Chain Reaction, Transduction, Gene Knockout, Marker

Journal: Scientific Reports

Article Title: Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells

doi: 10.1038/srep05105

Figure Lengend Snippet: Analyses of target gene knockout frequencies following AdV-mediated introduction of gRNA S1 :Cas9 and TALEN-L S1 :TALEN-R S1 complexes into hMSCs versus human myoblasts. (A) Target site genotyping assays. Upper and lower panels, agarose gel electrophoresis of AAVS1 -specific PCR products amplified from chromosomal DNA of myoblasts and hMSCs, respectively, co-transduced with the indicated MOIs of AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 (in TU/cell). Before loading the amplicons were denaturated, re-annealed and treated with the mismatch-sensitive enzyme T7EI. The cells were co-transduced at a combined MOI of 80 TU/cell with different ratios of AdV Δ2 P.Cas9.F 50 and AdV Δ2 U6.gRNA S1 .F 50 or with 1:1 mixtures of AdV Δ2 P.TALEN-L S1 .F 50 and AdV Δ2 P.TALEN-R S1 .F 50 . Mock-transduced cells or cells exposed to 80 TU/cell of AdV Δ2 P.Cas9.F 50 , AdV Δ2 U6.gRNA S1 .F 50 or AdV Δ2 P.TALEN-L S1 .F 50 served as negative controls. Solid and open arrowheads indicate the positions of undigested and T7EI-digested amplicons, respectively. Lanes M, Gene Ruler DNA Ladder marker. (B) Target gene knockout activities in hMSCs and myoblasts transduced with AdVs. Target gene disruption frequencies in cells co-transduced with AdV pairs AdV Δ2 P.Cas9.F 50 /AdV Δ2 U6.gRNA S1 .F 50 and AdV Δ2 P.TALEN-L S1 .F 50 /AdV Δ2 P.TALEN-R S1 .F 50 as determined by densitometry of DNA fragments depicted in Fig. 7A. (C) Gene delivery activities in hMSCs and myoblasts transduced with AdVs. Percentages of transduced cells incubated with different mixtures of reporter vector AdV Δ2 P.eGFP.F 50 and AdV Δ2 U6.gRNA S1 .F 50 , applied at a combined MOI of 80 TU/cell. Cells exposed only to AdV Δ2 U6.gRNA S1 .F 50 at an MOI of 80 TU/cell served as negative controls. The frequency of eGFP-positive cells was determined by flow cytometry at three days post-transduction. (D) Cumulative data corresponding to the gene knockout frequencies in hMSCs versus human myoblasts following AdV-mediated delivery of gRNA S1 :Cas9 complexes into these cells. Plotted data show the mean ± standard deviation (n = 7).

Article Snippet: One fifth of each PCR mixture was incubated in 15-μl reactions with 1 × NEBuffer 2 and 5 U of T7EI (both from New England Biolabs).

Techniques: Gene Knockout, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Transduction, Marker, Incubation, Plasmid Preparation, Flow Cytometry, Cytometry, Standard Deviation