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

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 modificati...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 113; no. 15; pp. 4122 - 4127
Main Authors van Ravesteyn, Thomas W., Dekker, Marleen, Fish, Alexander, Sixma, Titia K., Wolters, Astrid, Dekker, Rob J., te Riele, Hein P. J.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 12.04.2016
National Acad Sciences
SeriesFrom the Cover
Subjects
Animals
Base Pair Mismatch
Biological Sciences
Cells
Deoxyribonucleic acid
DNA
DNA - metabolism
DNA Breaks, Double-Stranded
DNA Mismatch Repair
DNA Repair
DNA, Single-Stranded
E coli
Escherichia coli
Escherichia coli - genetics
Genes
Genetics
Oligonucleotides - genetics
Rodents
locked nucleic acid
subtle gene modification
embryonic stem cells
DNA mismatch repair
single-stranded oligonucleotides
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Summary: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 evadesMMR 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.
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Author contributions: T.W.v.R., T.K.S., and H.P.J.t.R. designed research; T.W.v.R., M.D., A.F., A.W., and R.J.D. performed research; and T.W.v.R. and H.P.J.t.R. wrote the paper.
Edited by James E. Haber, Brandeis University, Waltham, MA, and approved January 6, 2016 (received for review July 7, 2015)
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1513315113