Format

Send to

Choose Destination
Cell Rep. 2016 Mar 8;14(9):2263-2272. doi: 10.1016/j.celrep.2016.02.018. Epub 2016 Feb 25.

Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases.

Author information

1
INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
2
INSERM U1064, CHU de Nantes, Nantes 44093, France; Platform Rat Transgenesis Immunophenomic, CNRS UMS3556, Nantes 44093, France.
3
Amagen, CNRS UMS 3504, INRA UMS 1374, Institut de Neurobiologie A. Fessard, Gif-sur-Yvette 91198, France.
4
genOway, Lyon 69007, France.
5
INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France. Electronic address: jpconcordet@mnhn.fr.
6
INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France. Electronic address: carine.giovannangeli@mnhn.fr.

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.

PMID:
26923600
DOI:
10.1016/j.celrep.2016.02.018
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center