title: Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases.
abstract: Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Renaud
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Boix
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
authors.forename: Marine
authors.initials: M
authors.lastname: Charpentier
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
authors.forename: Anne
authors.initials: A
authors.lastname: De Cian
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Cochennec
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Duvernois-Berthet
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
authors.forename: Loïc
authors.initials: L
authors.lastname: Perrouault
authors.affiliation: INSERM U1064, CHU de Nantes, Nantes 44093, France; Platform Rat Transgenesis Immunophenomic, CNRS UMS3556, Nantes 44093, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Tesson
authors.affiliation: Amagen, CNRS UMS 3504, INRA UMS 1374, Institut de Neurobiologie A. Fessard, Gif-sur-Yvette 91198, France.
authors.forename: Joanne
authors.initials: J
authors.lastname: Edouard
authors.affiliation: INSERM U1064, CHU de Nantes, Nantes 44093, France; Platform Rat Transgenesis Immunophenomic, CNRS UMS3556, Nantes 44093, France.
authors.forename: Reynald
authors.initials: R
authors.lastname: Thinard
authors.affiliation: genOway, Lyon 69007, France.
authors.forename: Yacine
authors.initials: Y
authors.lastname: Cherifi
authors.affiliation: INSERM U1064, CHU de Nantes, Nantes 44093, France; Platform Rat Transgenesis Immunophenomic, CNRS UMS3556, Nantes 44093, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Menoret
authors.affiliation: genOway, Lyon 69007, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Fontanière
authors.affiliation: Amagen, CNRS UMS 3504, INRA UMS 1374, Institut de Neurobiologie A. Fessard, Gif-sur-Yvette 91198, France.
authors.forename: Noémie
authors.initials: N
authors.lastname: de Crozé
authors.affiliation: genOway, Lyon 69007, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Fraichard
authors.affiliation: Amagen, CNRS UMS 3504, INRA UMS 1374, Institut de Neurobiologie A. Fessard, Gif-sur-Yvette 91198, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Sohm
authors.affiliation: INSERM U1064, CHU de Nantes, Nantes 44093, France; Platform Rat Transgenesis Immunophenomic, CNRS UMS3556, Nantes 44093, France.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Anegon
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France. Electronic address: jpconcordet@mnhn.fr.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Concordet
authors.affiliation: INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France. Electronic address: carine.giovannangeli@mnhn.fr.
authors.forename: Carine
authors.initials: C
authors.lastname: Giovannangeli
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D004720:Endonucleases / Q000235:genetics*; D000072669:Gene Editing; D018390:Gene Targeting; D006801:Humans; D054643:INDEL Mutation; D051379:Mice; D009841:Oligonucleotides / Q000235:genetics; D051381:Rats; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics*; D015027:Zebrafish
chemical_list: D009841:Oligonucleotides; D004720:Endonucleases; D000069896:Transcription Activator-Like Effector Nucleases