Format

Send to

Choose Destination
See comment in PubMed Commons below
Nucleic Acids Res. 2011 Sep 1;39(16):7336-47. doi: 10.1093/nar/gkr183. Epub 2011 May 23.

Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion.

Author information

1
Wyss Institute for Biologically Inspired Engineering, Harvard University, Department of Genetics, Harvard Medical School, Harvard University, Boston, MA 02115, USA. hhwang@genetics.med.harvard.edu

Abstract

Genome engineering using single-stranded oligonucleotides is an efficient method for generating small chromosomal and episomal modifications in a variety of host organisms. The efficiency of this allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. However, global MMR inactivation generally results in significant accumulation of undesired background mutations. Here, we present a novel strategy using oligos containing chemically modified bases (2'-Fluoro-Uridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine) in place of the standard T, C, A or G to avoid mismatch detection and repair, which we tested in Escherichia coli. This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5' and 3' termini of the oligo. These results further elucidate the mechanism of oligo-mediated allelic replacement (OMAR) and enable improved methodologies for efficient, large-scale engineering of genomes.

PMID:
21609953
PMCID:
PMC3167615
DOI:
10.1093/nar/gkr183
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Silverchair Information Systems Icon for PubMed Central
    Loading ...
    Support Center