Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases
2016
Summary
Genome editinghas now been reported in many systems using TALEN and
CRISPR-
Cas9nucleases. Precise mutations can be introduced during
homology-directed repairwith donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for
gene knockoutand optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified
oligonucleotidesstrongly enhances
genome editingefficiency of single-stranded
oligonucleotidedonors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified
oligonucleotidetoxicity, 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
indelsthat are asymmetrically positioned, consistent with
genome editingtaking place by two steps of single-strand annealing.
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