Effect of Tdt on meganuclease-induced mutagenesis.

<p>(A) Schematic representation of the transgene measuring NHEJ activity. A GFP gene lacking the ATG start codon was cloned out of frame and downstream of an exogenous sequence containing a meganuclease recognition site. (B) Quantification by flow cytometry of GFP positive cells 3 days post transfection with meganuclease alone (empty) or with meganuclease and Tdt (Tdt); experiments performed in triplicate. (C) Determination of TM of 2 independent experiments by sequence analysis of locus specific amplicons (454 Roche). On average, 10,000 amplicons were sequenced per experiment. Dark blue, insertion events; light blue, deletion events. The inset graph represents, as a function of insertion size, the percentage of meganuclease-induced TM with (green) or without (blue) Tdt. (D) Targeted mutagenesis at endogenous loci quantified by amplicon sequencing analysis. Meganucleases RAG1m, DMD21m (left panel) or CAPNS1m (right panel) were used either alone (empty) or with Tdt. Percentages of induced-TM are depicted as well the size of DNA insertions (inset graph). The mean percentage of insertion measured on the 3 endogenous loci is depicted.