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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.

journal_name

Cell Rep

journal_title

Cell reports

authors

Renaud JB,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 JP,Giovannangeli C

doi

10.1016/j.celrep.2016.02.018

subject

Has Abstract

pub_date

2016-03-08 00:00:00

pages

2263-2272

issue

9

issn

2211-1247

pii

S2211-1247(16)30104-8

journal_volume

14

pub_type

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