Dissociation solution (ReproCELL Incorporated, Japan), transferred on Geltrex (Life Technologies Corporation, USA) coated dishes and cultivated with MEF-conditioned stem cell medium. The day of transfection, cells were pre-incubated one hour with 10 mM Y-27632 Rock inhibitor. Electroporation was carried out with the Human Stem cells nucleofector solution 2 (Lonza group Ltd, Switzerland) using B-016 transfection program. Cells (16106) were transfected with 6 mg of CAPNS1 meganuclease coding plasmid (fused or not to scTrex endonuclease). A total of 10 mg ofMethods to Improve Targeted MutagenesisMethods to Improve Targeted MutagenesisFigure 1. Effect of Tdt on meganuclease-induced mutagenesis. (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. doi:10.1371/journal.pone.0053217.gDNA was used per transfection reaction. Cells were then seeded on Geltrex pre-coated 6-well plates and cultivated during 72 h in MEF-conditioned stem cell medium (changed daily) before being collected for genomic DNA extraction and amplicon sequencing analysis.Creating single-chain TREX2 (scTrex)A linker of 11 amino acids (TPPQTGLDVPY) was designed to bridge the C-terminal alanine of the N-terminal Trex2 molecule to the serine at the N-terminus of the second Trex2 molecule in the homodimer. To create the single-chain molecule, a strategy was adopted using a unique PstI restriction site within the Trex DNA sequence. Briefly, the Trex2 coding sequence was cloned into a mammalian expression vector (pcDNA3.1) and primers were designed to cover the PstI site (PstI_for/PstI_rev), along with primers encompassing either a region of the N-terminal Trex sequence and the linker (Trex2Link_for); or part of the C-terminal Trex2 sequence plus the linker (Trex2Link_rev). Two independent PCR’s were carried out creating two products for use as template in an MedChemExpress SIS-3 assembly PCR using the PstI_for and PstI_rev primers. The resulting product was digested by PstI and ligated into the vector containing Trex2, also digested by the same enzyme, creating the single-chain Trex2 with the 11 amino acid linker.Fusing scTrex to a meganucleaseTo create scTrex-meganuclease fusions, we first fused Trex2 to a meganuclease at its N-terminus, using a ten amino acid glycineserine stretch (GGGGS)2 as a linker. Fusion protein constructs were 298690-60-5 web obtained by separately amplifying the two ORFs using primer pairs Link10MNRev and CMV_for (59CGCA.Dissociation solution (ReproCELL Incorporated, Japan), transferred on Geltrex (Life Technologies Corporation, USA) coated dishes and cultivated with MEF-conditioned stem cell medium. The day of transfection, cells were pre-incubated one hour with 10 mM Y-27632 Rock inhibitor. Electroporation was carried out with the Human Stem cells nucleofector solution 2 (Lonza group Ltd, Switzerland) using B-016 transfection program. Cells (16106) were transfected with 6 mg of CAPNS1 meganuclease coding plasmid (fused or not to scTrex endonuclease). A total of 10 mg ofMethods to Improve Targeted MutagenesisMethods to Improve Targeted MutagenesisFigure 1. Effect of Tdt on meganuclease-induced mutagenesis. (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. doi:10.1371/journal.pone.0053217.gDNA was used per transfection reaction. Cells were then seeded on Geltrex pre-coated 6-well plates and cultivated during 72 h in MEF-conditioned stem cell medium (changed daily) before being collected for genomic DNA extraction and amplicon sequencing analysis.Creating single-chain TREX2 (scTrex)A linker of 11 amino acids (TPPQTGLDVPY) was designed to bridge the C-terminal alanine of the N-terminal Trex2 molecule to the serine at the N-terminus of the second Trex2 molecule in the homodimer. To create the single-chain molecule, a strategy was adopted using a unique PstI restriction site within the Trex DNA sequence. Briefly, the Trex2 coding sequence was cloned into a mammalian expression vector (pcDNA3.1) and primers were designed to cover the PstI site (PstI_for/PstI_rev), along with primers encompassing either a region of the N-terminal Trex sequence and the linker (Trex2Link_for); or part of the C-terminal Trex2 sequence plus the linker (Trex2Link_rev). Two independent PCR’s were carried out creating two products for use as template in an assembly PCR using the PstI_for and PstI_rev primers. The resulting product was digested by PstI and ligated into the vector containing Trex2, also digested by the same enzyme, creating the single-chain Trex2 with the 11 amino acid linker.Fusing scTrex to a meganucleaseTo create scTrex-meganuclease fusions, we first fused Trex2 to a meganuclease at its N-terminus, using a ten amino acid glycineserine stretch (GGGGS)2 as a linker. Fusion protein constructs were obtained by separately amplifying the two ORFs using primer pairs Link10MNRev and CMV_for (59CGCA.