title: Use of internally nuclease-protected single-strand DNA oligonucleotides and silencing of the mismatch repair protein, MSH2, enhances the replication of corrected cells following gene editing.
abstract: BACKGROUND
  Gene editing is potentially a powerful technology for introducing genetic changes by using short single-stranded DNA oligonucleotides (ssODNs). However, their efficiency is reduced by the mismatch repair system, especially MSH2, which may suppress gene editing, although findings vary depending on readout and type of oligonucleotide used. Additionally, successfully edited cells are reported to arrest at the S- or G2-phase. In the present study, we evaluate whether a novel ssODN design and down-regulation of MSH2 expression allows the isolation of replicating gene-edited cells.
  
  
  METHODS
  Cultured Chinese hamster ovary cells expressing mutated enhanced green fluorescent protein were targeted with ssODNs of varying design, all capable of restoring fluorescence, which allows the monitoring of correction events by flow cytometry. Converted cells were isolated by cell sorting and grown to determine colony formation efficiencies. MSH2 expression was suppressed with small interfering RNA and the cell cycle distribution of cells transfected with ssODN was quantified by flow cytometry, following propidium iodide or DRAQ5 staining.
  
  
  RESULTS
  Although efficiency was higher using ssODN end-protected with phosphorothioate, the potential of edited cells to form colonies was lower than those targeted with unmodified ssODN. We established that ssODN transfection itself perturbs the cell cycle and that MSH2 gene silencing increases correction efficiency. In both cases, however, the effect was dependent on the positioning of the protected nucleotides. Importantly, when internally protected ssODN was used in combination with MSH2 suppression, a higher proportion of G1-phase corrected cells was observed 48-64 h after transfection.
  
  
  CONCLUSIONS
  Use of internally protected ssODN and downregulating cellular MSH2 activity may facilitate isolation of viable, actively replicating gene-edited cells.
authors.affiliation: Department of Medicine, Royal Free and University College Medical School, Royal Free Campus, London, UK.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Papaioannou
authors.forename: Petra
authors.initials: P
authors.lastname: Disterer
authors.forename: James S
authors.initials: JS
authors.lastname: Owen
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002453:Cell Cycle / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D053843:DNA Mismatch Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics*; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051718:MutS Homolog 2 Protein