Nsgenic locus in presence of Tdt. Sequences marked with asterisk (*) correspond to insertion events independent of the TDT activity. They are also found in the sample corresponding to cells transfected with meganuclease in absence of TDT. (DOC) Table S2 Targeted mutagenesis data. Nucleases encoding plasmids were transfected with or without DNA-end processing enzyme encoding plasmids in 10 mg of total DNA. Cells were harvested 3 days post-transfection for genomic DNA extraction and locus specific PCR amplification for deep sequencing analysis. Several thousands of sequences were obtained per PCR product and then analyzed for site-specific insertion or MedChemExpress TA-01 Deletion events. (DOC)proteins used in this study. scTrex2: Single chain molecule of Trex2 exonuclease. Two Trex2 monomers were fused together using the linker 1 : TPPQTGLDVPY. ScTrex-meganuclease: meganuclease results from the fusion of two engineered monomers linked by linker 2.. The single chain meganuclease was then fused by its N-t domain to the single chain Trex2 using the linker 3. The resulting molecule harbors endonuclease and 39-.59exonuclease activity. (DOCX)Figure S1 Deletion pattern induced 25331948 by MN in the presence of Tdt. Deletion events induced by RAG1m (A) DMD21m (B) or CAPNS1m (C) in the presence (green) or absence (blue) of Tdt were analyzed. Deletion sizes are represented as percentage of total deletion events. Note that CAPNS1m induces large and diverse deletions that were analyzed by deletion classes (range of deletions size). (EPS) Figure S2 Targeted mutagenesis pattern induced by MNs in the presence of Trex or scTrex. Percentages of TM events induced by RAG1m (A) DMD21m (B) or CAPNS1m (C) with or without (empty) Trex or scTrex are shown. Del2, 2 bp deletion; Del3, 3 bp deletion; Del4, 4 bp deletion; Other, more than 4 bp Deletions as well as other TM events induced byAcknowledgmentsWe thank the Cardiovascular Research Center (Mount Sinai School of Medicine, York, USA) for iPS cells.Author ContributionsConceived and designed the experiments: F. Delacote CP RM GS PD F. ^ Daboussi. Performed the experiments: F. Delacote CP VG MD CR NO ^ RM F. Daboussi. Analyzed the data: GS F. Daboussi PD FP. Contributed reagents/materials/analysis tools: RM MD. Wrote the paper: F. Delacote ^ CP PD FP.Methods to Improve Targeted Mutagenesis
CP21 site alterations of the sodium current (INa) in the human heart can lead to diseases responsible for cardiac arrhythmias, such as Brugada Syndrome (BrS) [1]. This syndrome, first described in 1992, is characterized by the presence of ST segment elevation in the right precordial leads (V1 3) of the electrocardiogram (ECG), without major structural alterations in the heart [2]. The prevalence of BrS is in the range of 1? in every 10,000 individuals and is an important cause of Sudden Cardiac Death (SCD) [3]. Since the discovery of the first genetic variation in the cardiac sodium channel gene, SCN5A, associated with BrS [4], many studies have classified this syndrome as a genetic disease with autosomal dominant inheritance and incomplete penetrance [5]. It has been demonstrated that mutations in SCN5A associated with BrS result in loss-of-function of the current carried by the cardiac type sodium channel (Nav1.5) [6]. Different mechanisms are known to produce channel loss-of-function, including reduced expression of the channel in the plasma membrane, changes in the voltage dependence of the channel activation or inactivation, or altered channel kinetics [7]. In.Nsgenic locus in presence of Tdt. Sequences marked with asterisk (*) correspond to insertion events independent of the TDT activity. They are also found in the sample corresponding to cells transfected with meganuclease in absence of TDT. (DOC) Table S2 Targeted mutagenesis data. Nucleases encoding plasmids were transfected with or without DNA-end processing enzyme encoding plasmids in 10 mg of total DNA. Cells were harvested 3 days post-transfection for genomic DNA extraction and locus specific PCR amplification for deep sequencing analysis. Several thousands of sequences were obtained per PCR product and then analyzed for site-specific insertion or deletion events. (DOC)proteins used in this study. scTrex2: Single chain molecule of Trex2 exonuclease. Two Trex2 monomers were fused together using the linker 1 : TPPQTGLDVPY. ScTrex-meganuclease: meganuclease results from the fusion of two engineered monomers linked by linker 2.. The single chain meganuclease was then fused by its N-t domain to the single chain Trex2 using the linker 3. The resulting molecule harbors endonuclease and 39-.59exonuclease activity. (DOCX)Figure S1 Deletion pattern induced 25331948 by MN in the presence of Tdt. Deletion events induced by RAG1m (A) DMD21m (B) or CAPNS1m (C) in the presence (green) or absence (blue) of Tdt were analyzed. Deletion sizes are represented as percentage of total deletion events. Note that CAPNS1m induces large and diverse deletions that were analyzed by deletion classes (range of deletions size). (EPS) Figure S2 Targeted mutagenesis pattern induced by MNs in the presence of Trex or scTrex. Percentages of TM events induced by RAG1m (A) DMD21m (B) or CAPNS1m (C) with or without (empty) Trex or scTrex are shown. Del2, 2 bp deletion; Del3, 3 bp deletion; Del4, 4 bp deletion; Other, more than 4 bp Deletions as well as other TM events induced byAcknowledgmentsWe thank the Cardiovascular Research Center (Mount Sinai School of Medicine, York, USA) for iPS cells.Author ContributionsConceived and designed the experiments: F. Delacote CP RM GS PD F. ^ Daboussi. Performed the experiments: F. Delacote CP VG MD CR NO ^ RM F. Daboussi. Analyzed the data: GS F. Daboussi PD FP. Contributed reagents/materials/analysis tools: RM MD. Wrote the paper: F. Delacote ^ CP PD FP.Methods to Improve Targeted Mutagenesis
Alterations of the sodium current (INa) in the human heart can lead to diseases responsible for cardiac arrhythmias, such as Brugada Syndrome (BrS) [1]. This syndrome, first described in 1992, is characterized by the presence of ST segment elevation in the right precordial leads (V1 3) of the electrocardiogram (ECG), without major structural alterations in the heart [2]. The prevalence of BrS is in the range of 1? in every 10,000 individuals and is an important cause of Sudden Cardiac Death (SCD) [3]. Since the discovery of the first genetic variation in the cardiac sodium channel gene, SCN5A, associated with BrS [4], many studies have classified this syndrome as a genetic disease with autosomal dominant inheritance and incomplete penetrance [5]. It has been demonstrated that mutations in SCN5A associated with BrS result in loss-of-function of the current carried by the cardiac type sodium channel (Nav1.5) [6]. Different mechanisms are known to produce channel loss-of-function, including reduced expression of the channel in the plasma membrane, changes in the voltage dependence of the channel activation or inactivation, or altered channel kinetics [7]. In.
