LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

Proc Natl Acad Sci U S A. 2016 Apr 12;113(15):4122-7. doi: 10.1073/pnas.1513315113. Epub 2016 Mar 7.

Abstract

Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.

Keywords: DNA mismatch repair; embryonic stem cells; locked nucleic acid; single-stranded oligonucleotides; subtle gene modification.

Publication types

  • Research Support, Non-U.S. Gov't
  • Comment

MeSH terms

  • Animals
  • Base Pair Mismatch
  • DNA / metabolism
  • DNA Breaks, Double-Stranded
  • DNA Mismatch Repair
  • DNA Repair
  • DNA, Single-Stranded*
  • Escherichia coli / genetics*
  • Oligonucleotides / genetics

Substances

  • DNA, Single-Stranded
  • Oligonucleotides
  • DNA