E-specific insertion or deletion events (Table S2).Extrachromosomal activity and survival

E-specific insertion or deletion events (Table S2).Extrachromosomal activity and survival assays in CHO-KActivity and toxicity in mammalian cells was measured as previously reported by Grizot et al. [31] For extrachromosomal assays, CHO-K1 cells were seeded in 96-well plates at 2,500 cells per well and transfected one day post platting with 150 or 200 ng 25033180 of total DNA using Polyfect transfection reagent according to the supplier’s protocol (Qiagen). In the survival assay, 10 ng of GFPencoding plasmid was mixed with various amounts (from 2.5 to 25 ng) of meganuclease expression vectors.NHEJ cellular modelThe construct monitoring NHEJ is made of an ATG start codon followed by (i) the HA tag sequence; (ii) a GS meganuclease (GSm) recognition site; (iii) a glycine-serine stretch, and; (iv) a GFP reporter gene lacking the start codon. In this arrangement the GFP gene is inactive due to a frame-shift introduced by the GS recognition site, however, creation of a DNA DSB by GSm, followed by a mutagenic NHEJ repair event, can lead to restoration of GFP gene expression in frame with the ATG start codon. This sequence was cloned into the human RAG1 endogenous locus using the plasmid hsRAG1 Integration Matrix CMV Neo from cGPSH Custom Human Full Kit DD (Cellectis Bioresearch). The plasmid contains all the necessary components to obtain by homologous recombination a highly efficient insertion event of the transgene at the RAG1 endogenous locus. It is composed of two homology arms of 1.8 and 1.2 kbp separated by an expression cassette of the neomycin resistance gene driven by a mammalian CMV promoter and our transgene under a second CMV promoter. It also contains the HSV-TK gene under EF1a promoter control placed outside the homology arms. This plasmid was transfected in 293H cells and clones presenting double resistance (neomycin and ganciclovir) were used to quantify NHEJ induced by GSm.Transfection in Detroit 551 cells to monitor meganuclease-induced mutagenesis at CAPNS1 locusElectroporation was carried out with the NHDF nucleofactor kit and device (Lonza group Ltd, Switzerland) under the U-020 transfection program. Cells (16106) were transfected with 6 mg of CAPNS1 meganuclease coding plasmid (fused or not to scTrex endonuclease) and 4 mg of Tdt, scTrex or pUC. A total of 10 mg of DNA was used per transfection reaction. Cells were then plated in 6-well plates and cultivated during 72 h before to be collected for genomic DNA 223488-57-1 extraction and amplicon sequencing analysis.Transfection in the NHEJ cellular modelOne million cells were seeded one day prior to transfection. These cells were co-transfected with 3 mg of plasmid encoding GSm or scTrex-GSm, with or without 2 mg of plasmid encoding Tdt, Trex or scTrex in 5 mg of total DNA by complementation with a pUC vector using 25 ml of lipofectamine (Invitrogen) according to the manufacturer’s instructions. Four days posttransfection, cells were buy 94-09-7 harvested and the percentage of GFPpositive cells was measured by flow cytometry analysis using Guava instrumentation (Millipore). Genomic DNA was extracted from cell population and locus specific PCRs were performed using the following primers: 59-CCATCTTransfection in iPS cells to monitor meganucleaseinduced mutagenesis at CAPNS1 locusTwo days before transfection, cells were treated with CDK dissociation solution (ReproCELL Incorporated, Japan), transferred on Geltrex (Life Technologies Corporation, USA) coated dishes and cultivated with MEF-conditioned stem cell.E-specific insertion or deletion events (Table S2).Extrachromosomal activity and survival assays in CHO-KActivity and toxicity in mammalian cells was measured as previously reported by Grizot et al. [31] For extrachromosomal assays, CHO-K1 cells were seeded in 96-well plates at 2,500 cells per well and transfected one day post platting with 150 or 200 ng 25033180 of total DNA using Polyfect transfection reagent according to the supplier’s protocol (Qiagen). In the survival assay, 10 ng of GFPencoding plasmid was mixed with various amounts (from 2.5 to 25 ng) of meganuclease expression vectors.NHEJ cellular modelThe construct monitoring NHEJ is made of an ATG start codon followed by (i) the HA tag sequence; (ii) a GS meganuclease (GSm) recognition site; (iii) a glycine-serine stretch, and; (iv) a GFP reporter gene lacking the start codon. In this arrangement the GFP gene is inactive due to a frame-shift introduced by the GS recognition site, however, creation of a DNA DSB by GSm, followed by a mutagenic NHEJ repair event, can lead to restoration of GFP gene expression in frame with the ATG start codon. This sequence was cloned into the human RAG1 endogenous locus using the plasmid hsRAG1 Integration Matrix CMV Neo from cGPSH Custom Human Full Kit DD (Cellectis Bioresearch). The plasmid contains all the necessary components to obtain by homologous recombination a highly efficient insertion event of the transgene at the RAG1 endogenous locus. It is composed of two homology arms of 1.8 and 1.2 kbp separated by an expression cassette of the neomycin resistance gene driven by a mammalian CMV promoter and our transgene under a second CMV promoter. It also contains the HSV-TK gene under EF1a promoter control placed outside the homology arms. This plasmid was transfected in 293H cells and clones presenting double resistance (neomycin and ganciclovir) were used to quantify NHEJ induced by GSm.Transfection in Detroit 551 cells to monitor meganuclease-induced mutagenesis at CAPNS1 locusElectroporation was carried out with the NHDF nucleofactor kit and device (Lonza group Ltd, Switzerland) under the U-020 transfection program. Cells (16106) were transfected with 6 mg of CAPNS1 meganuclease coding plasmid (fused or not to scTrex endonuclease) and 4 mg of Tdt, scTrex or pUC. A total of 10 mg of DNA was used per transfection reaction. Cells were then plated in 6-well plates and cultivated during 72 h before to be collected for genomic DNA extraction and amplicon sequencing analysis.Transfection in the NHEJ cellular modelOne million cells were seeded one day prior to transfection. These cells were co-transfected with 3 mg of plasmid encoding GSm or scTrex-GSm, with or without 2 mg of plasmid encoding Tdt, Trex or scTrex in 5 mg of total DNA by complementation with a pUC vector using 25 ml of lipofectamine (Invitrogen) according to the manufacturer’s instructions. Four days posttransfection, cells were harvested and the percentage of GFPpositive cells was measured by flow cytometry analysis using Guava instrumentation (Millipore). Genomic DNA was extracted from cell population and locus specific PCRs were performed using the following primers: 59-CCATCTTransfection in iPS cells to monitor meganucleaseinduced mutagenesis at CAPNS1 locusTwo days before transfection, cells were treated with CDK dissociation solution (ReproCELL Incorporated, Japan), transferred on Geltrex (Life Technologies Corporation, USA) coated dishes and cultivated with MEF-conditioned stem cell.

Leave a Reply