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Fig. 2 | Biology Direct

Fig. 2

From: Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells

Fig. 2

Comparison of efficiencies of the Cpf1 nucleases when used with either a PCR- or a plasmid-derived crRNA in N2a mouse neuroblastoma cells. a Scheme of a plasmid template for nuclease expression (orange: Cpf1 expression cassette) and of a PCR-based template for crRNA expression [a human U6 promoter PCR product (blue with scarlet forward and reverse primers) with a hybridized dsDNA oligonucleotide (light blue)]. b Scheme of a dual expression plasmid template for both the nuclease (orange) and the crRNA (light blue) with the human U6 promoter (blue arrow). c Percentages of GFP positive cells formed above the background level, induced by the nuclease action of either AsCpf1 or LbCpf1. N2a cells are cotransfected with either a PCR-based template for crRNA and a plasmid for Cpf1 nuclease expression (as depicted on Fig. 2a), labelled as “PCR”, or with a dual expression plasmid for both the nuclease and the crRNA (as depicted on Fig. 2b), labelled as “plasmid”, respectively. GFP positive cells are counted two days after transfection by flow cytometry. All samples are also cotransfected by an mCherry expression vector as a transfection reference and the GFP expression is analysed within the mCherry positive population. The transfection efficiencies measured based on the fluorescence of mCherry ranged within 55.9 % ± 10.5 % when the plasmid was used, whereas 45.0 % ± 8.4 %, when the PCR product. As negative control, a crRNA-less, active AsCpf1 nuclease expression vector was used for each target and the obtained levels of GFP fluorescence for these controls are subtracted from the corresponding samples’ values. Three parallel transfections were made for each case. Error bars show the mean ± standard deviation of percentages measured in N = 3 independent experiments. Plasmid-expression of the crRNA results in 2- to 4-fold increase in GFP positive cells as compared to a PCR-template expression

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