| First Author | van Ravesteyn TW | Year | 2016 |
| Journal | Proc Natl Acad Sci U S A | Volume | 113 |
| Issue | 15 | Pages | 4122-7 |
| PubMed ID | 26951689 | Mgi Jnum | J:231916 |
| Mgi Id | MGI:5775498 | Doi | 10.1073/pnas.1513315113 |
| Citation | van Ravesteyn TW, et al. (2016) LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells. Proc Natl Acad Sci U S A 113(15):4122-4127 |
| abstractText | 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. |
