Effect of Trex2, scTrex or scTrex fused to meganuclease on meganuclease-induced mutagenesis.

<p>(A) Quantification by flow cytometry of the percentage of GFP positive cells 3 days post transfection with meganuclease alone (empty), with meganuclease and Trex2 or scTrex and meganuclease fused to scTrex; experiments performed in triplicate. (B) Determination of meganuclease-induced TM by sequence analysis of locus specific amplicons in the presence of wild-type Trex2 (Trex2), the engineered single-chain variant (scTrex) or meganuclease fused to scTrex. The inset graph shows the percentage of 2, 3, and 4 nucleotide (Del2, Del3, Del4) deletions among all TM events. E, meganuclease alone; T, meganuclease with Trex2; scT, meganuclease with scTrex. (C) Targeted mutagenesis at endogenous loci quantified by amplicon sequencing analysis. On average 10,000 amplicons were sequenced per experiment. Percentage of TM induced by meganucleases RAG1m, DMD21m (left panel) or CAPNS1m (right panel) are depicted. Empty, meganuclease alone; Trex2, meganuclease with wild-type Trex2. (D) scTrex was fused, respectively, to meganucleases targeting the hCAPNS1 and hRAG1 genes, and TM was determined by sequence analysis of locus specific amplicons.