ZFIN ID: ZDB-PUB-170214-34
Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases
Renaud, J.B., Boix, C., Charpentier, M., De Cian, A., Cochennec, J., Duvernois-Berthet, E., Perrouault, L., Tesson, L., Edouard, J., Thinard, R., Cherifi, Y., Menoret, S., Fontanière, S., de Crozé, N., Fraichard, A., Sohm, F., Anegon, I., Concordet, J.P., Giovannangeli, C.
Date: 2016
Source: Cell Reports   14: 2263-72 (Journal)
Registered Authors: Sohm, Frédéric
Keywords: none
MeSH Terms:
  • Animals
  • Base Sequence
  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Endonucleases/genetics*
  • Gene Editing*
  • Gene Targeting
  • Humans
  • INDEL Mutation
  • Mice
  • Oligonucleotides/genetics
  • Rats
  • Transcription Activator-Like Effector Nucleases/genetics*
  • Zebrafish
PubMed: 26923600 Full text @ Cell Rep.
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.