Journal: Foods

Article Title: A Rapid and Visual Method for Nucleic Acid Detection of Escherichia coli O157:H7 Based on CRISPR/Cas12a-PMNT

doi: 10.3390/foods12020236

Figure Lengend Snippet: Feasibility of Cas12a–PMNT-based visual method for nucleic acid detection (1) NEBuffer + positive plasmid DNA + Cas12a + Rnase Inhibitor + ssDNA + PMNT; (2) NEBuffer + positive plasmid DNA + crRNA + Rnase Inhibitor + ssDNA + PMNT; (3) NEBuffer + crRNA + Cas12a + Rnase Inhibitor + ssDNA + PMNT; (4) NEBuffer + positive plasmid DNA + crRNA + Cas12a + Rnase Inhibitor + PMNT; (5) NEBuffer + ssDNA + PMNT; (6) NEBuffer + positive plasmid DNA + crRNA + Cas12a + Rnase Inhibitor + ssDNA + PMNT; (7) NEBuffer + negative plasmid DNA + crRNA + Cas12a + Rnase Inhibitor + ssDNA + PMNT; (8) S1 Reaction Buffer + S1 Nuclease + ssDNA + PMNT.

Article Snippet: EnGen Lba Cas12a (Cpf1) kit for Cas12a-mediated target cleavage assay was purchased from New England Biolabs Inc. (Ipswich, MA, USA).

Techniques: Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

doi: 10.3390/ijms232416218

Figure Lengend Snippet: Single guide RNA sequences and primers used for amplification and sequencing of target genomic regions.

Article Snippet: The laboratory synthesis of the sgRNA was carried out by mixing the target-specific oligos with the manufacturer’s (New England Biolabs, Hitchin, UK) reaction mix, comprising nuclease-free water (2 μL); EnGen 2× sgRNA reaction mix (dNTPs, S. pyogenes Cas9 scaffold oligo) (10 μL); target-specific DNA oligo (1 μM; 5 μL); DTT (0.1 M; 1 μL); and EnGen sgRNA Enzyme mix (2 μL).

Techniques: Amplification, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

doi: 10.3390/ijms232416218

Figure Lengend Snippet: sgRNA analysis and in vitro cleavage assay for cr2 and mmp9 genes. ( a ) sgRNA analysis on 2% agarose gel. UD: undenatured sgRNA, D: denatured sgRNA. Single bands detected for the denatured sgRNAs highlighted the purity of the products, and multiple bands in the undenatured sgRNA resulted from the presence of RNA secondary structures; ( b ) gel shows duplicate lanes for each sample. Lanes 1–6: experimental samples, lane 7: no Cas9 control, lane 8: no sgRNA control; ( c ) lane 1: no Cas9 control, lane 2: no sgRNA control, lanes 3–5: experimental samples. Cleavage products at expected band sizes were detected for all samples containing the sgRNA + Cas9 + gDNA. No product at the expected band size was observed for all the control samples.

Article Snippet: The laboratory synthesis of the sgRNA was carried out by mixing the target-specific oligos with the manufacturer’s (New England Biolabs, Hitchin, UK) reaction mix, comprising nuclease-free water (2 μL); EnGen 2× sgRNA reaction mix (dNTPs, S. pyogenes Cas9 scaffold oligo) (10 μL); target-specific DNA oligo (1 μM; 5 μL); DTT (0.1 M; 1 μL); and EnGen sgRNA Enzyme mix (2 μL).

Techniques: In Vitro, Cleavage Assay, Agarose Gel Electrophoresis

Journal: International Journal of Molecular Sciences

Article Title: CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

doi: 10.3390/ijms232416218

Figure Lengend Snippet: Ribonucleoprotein (RNP) and plasmid (px458) transfection of ( a ) ASK-1 and ( b ) CHSE-214 cells. Vertical panels show cells transfected with (i) RNP (sgRNA:Cas9-EGFP complex); (ii) Cas9-EGFP protein control; (iii) plasmid (px458); (iv) sham control. Horizontal panels show pictures of cells taken with (1) fluorescent channel; (2) light channel; (3) overlay of the light and fluorescent panels. Pictures were taken at day 7 after RNP electroporation (1600 V, 10 ms, 3 pulses) and day 2 after px458 electroporation (1200 V, 20 ms, 2 pulses). Strong adherence of Cas9-EGFP was observed on the cell surface of CHSE-214, and it was difficult to entirely wash off with PBS, explaining the high background fluorescence in the Cas9-EGFP- and RNP-transfected CHSE-214 cells. Magnification X20. (Fluorescent pictures of transfected SHK-1 cells are not shown).

Article Snippet: The laboratory synthesis of the sgRNA was carried out by mixing the target-specific oligos with the manufacturer’s (New England Biolabs, Hitchin, UK) reaction mix, comprising nuclease-free water (2 μL); EnGen 2× sgRNA reaction mix (dNTPs, S. pyogenes Cas9 scaffold oligo) (10 μL); target-specific DNA oligo (1 μM; 5 μL); DTT (0.1 M; 1 μL); and EnGen sgRNA Enzyme mix (2 μL).

Techniques: Plasmid Preparation, Transfection, Electroporation, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

doi: 10.3390/ijms232416218

Figure Lengend Snippet: Analyses of mutations in the target regions of cr2 and mmp9 genes due to the applied CRISPR/Cas9 strategies. ( a ) T7E1 assay of the cr2 RNP-transfected CHSE-214 cells. Cleaved fragments corresponding to T7 endonuclease 1 digestion of gDNA extract from the RNP-transfected cells are indicated with white arrows in lanes 3 and 4. Similar results were obtained in RNP-transfected ASK-1 and SHK-1 cells for both cr2 and mmp9 . ( b ) Representative Sanger sequencing chromatogram for the target region of cr2 (upper left) and mmp9 (upper right) in ASK-1 cells, either wild-type (WT) or edited with CRISPR/Cas9. The binding regions are represented by the sgRNA rectangular bar, and the edited regions are boxed in dashed lines. ( c ) Representative output of DECODRE analysis of a cr2 RNP-transfected- (lower left) and an mmp9 RNP-transfected (lower right) ASK-1 cell. Indel type and editing frequency are indicated, as well as the aligned sequences of the wild-type and edited samples. The sgRNA including the PAM sequences (red and green bar) highlights the exact target sequence and Cas9 cut site. Boxed dashed lines indicate the exact site of the observed indel mutations.

Article Snippet: The laboratory synthesis of the sgRNA was carried out by mixing the target-specific oligos with the manufacturer’s (New England Biolabs, Hitchin, UK) reaction mix, comprising nuclease-free water (2 μL); EnGen 2× sgRNA reaction mix (dNTPs, S. pyogenes Cas9 scaffold oligo) (10 μL); target-specific DNA oligo (1 μM; 5 μL); DTT (0.1 M; 1 μL); and EnGen sgRNA Enzyme mix (2 μL).

Techniques: CRISPR, Transfection, Sequencing, Binding Assay