Journal: PLoS ONE

Article Title: No evidence of genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells

doi: 10.1371/journal.pone.0177444

Figure Lengend Snippet: A . EGFP-positive cells were observed in SaCas9 but not NgAgo transfected reporter cells. sgRNA for SaCas9 and 5’ phosphorylated single-strand DNA guide oligo for NgAgo both targeted human HBB . A single-strand DNA guide oligo was included in the SaCas9 transfection to equate transfection conditions and exclude possible inhibition of plasmid DNA transfection. B . Plasmid DNA and single-strand oligo DNA transfected into reporter cells. Cells were co-transfected with DNA expressing mCherry-MEX3C 659AA (a cytoplasmic protein, pseudocolored red) and Alexa488-labeled oligo targeting luciferase cDNA (pseudocolored green). Nucleus was stained by DAPI (pseudocolored blue). Scale bar: 50 μm.

Article Snippet: Then pSin-EF2- IL2RG - HBB -GFP Plasmid DNA was co-transfected with psPAX2 and pMD2G DNA (9, 6 and 3 μg respectively) into the cells mediated by 54 μl Fugene 6 according to the instructions of the manufacturer ( https://www.promega.com/-/media/files/resources/protocols/technical-manuals/101/fugene-6-transfection-reagent-protocol.pdf ).

Techniques: Transfection, Inhibition, Plasmid Preparation, Expressing, Labeling, Luciferase, Staining

Journal: PLoS ONE

Article Title: No evidence of genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells

doi: 10.1371/journal.pone.0177444

Figure Lengend Snippet: A . Untagged NgAgo produced green fluorescence positive cells independent of guide DNA. SaCas9, tagged NgAgo or untagged NgAgo were cotransfected with reporter plasmid DNA into HEK293T cells. In SaCas9 transfection single strand guide DNA was included to equate transfection conditions. Scale bar: 100 μm. B . Next-generation sequencing of the target region of reporter plasmid DNA amplified from cells co-transfected with untagged NgAgo and F2+R1 oligos. The most appearing seven types of reads are listed with their percentages. Positions of the guide oligos are highlighted, with direction indicated by arrows. C . Sequencing of the target region of human MYC promoter amplified from cells co-transfected with untagged NgAgo and the indicated oligos. The most appearing seven types of reads are listed with their percentages. Positions of the guide oligos are highlighted, with direction indicated by arrows.

Article Snippet: Then pSin-EF2- IL2RG - HBB -GFP Plasmid DNA was co-transfected with psPAX2 and pMD2G DNA (9, 6 and 3 μg respectively) into the cells mediated by 54 μl Fugene 6 according to the instructions of the manufacturer ( https://www.promega.com/-/media/files/resources/protocols/technical-manuals/101/fugene-6-transfection-reagent-protocol.pdf ).

Techniques: Produced, Fluorescence, Plasmid Preparation, Transfection, Next-Generation Sequencing, Amplification, Sequencing