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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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2016-04, Vol.113 (15), p.4122-4127 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | van Ravesteyn, Thomas W. Dekker, Marleen Fish, Alexander Sixma, Titia K. Wolters, Astrid Dekker, Rob J. te Riele, Hein P. J. |
| description | 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. |
| doi_str_mv | 10.1073/pnas.1513315113 |
| format | Article |
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J.</creator><creatorcontrib>van Ravesteyn, Thomas W. ; Dekker, Marleen ; Fish, Alexander ; Sixma, Titia K. ; Wolters, Astrid ; Dekker, Rob J. ; te Riele, Hein P. J.</creatorcontrib><description>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.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.1513315113</identifier><identifier>PMID: 26951689</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>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</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2016-04, Vol.113 (15), p.4122-4127</ispartof><rights>Volumes 1–89 and 106–113, copyright as a collective work only; author(s) retains copyright to individual articles</rights><rights>Copyright National Academy of Sciences Apr 12, 2016</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c500t-569adb3697dcf2918aea24e0c65c0f0e39c11d1e73dd2d258a63ee5c3c88d2f83</citedby><cites>FETCH-LOGICAL-c500t-569adb3697dcf2918aea24e0c65c0f0e39c11d1e73dd2d258a63ee5c3c88d2f83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/113/15.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/26469267$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/26469267$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,723,776,780,799,881,27901,27902,53766,53768,57992,58225</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26951689$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>van Ravesteyn, Thomas W.</creatorcontrib><creatorcontrib>Dekker, Marleen</creatorcontrib><creatorcontrib>Fish, Alexander</creatorcontrib><creatorcontrib>Sixma, Titia K.</creatorcontrib><creatorcontrib>Wolters, Astrid</creatorcontrib><creatorcontrib>Dekker, Rob J.</creatorcontrib><creatorcontrib>te Riele, Hein P. J.</creatorcontrib><title>LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>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.</description><subject>Animals</subject><subject>Base Pair Mismatch</subject><subject>Biological Sciences</subject><subject>Cells</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA - metabolism</subject><subject>DNA Breaks, Double-Stranded</subject><subject>DNA Mismatch Repair</subject><subject>DNA Repair</subject><subject>DNA, Single-Stranded</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Genes</subject><subject>Genetics</subject><subject>Oligonucleotides - genetics</subject><subject>Rodents</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkruO1DAUQCMEYoeFmgqIREOTXb9jN0ir5SmNoIHa8tg3sx459mAnICQ-HkczDLtUNHbhc4_vq2meYnSBUU8v99GUC8wxpfXA9F6zwkjhTjCF7jcrhEjfSUbYWfOolB1CSHGJHjZnRCiOhVSr5tf601U7JucHb83kU2zT0BYftwG6MmUTHbj2TWVS8NsUZxsgTd5BaU0I6Udpy7yZArRbiHDX42M7-jKayd50GfbG526fU332EKfWQgjlcfNgMKHAk-N93nx99_bL9Ydu_fn9x-urdWc5QlPHhTJuQ4XqnR2IwtKAIQyQFdyiAQFVFmOHoafOEUe4NIICcEutlI4Mkp43rw_e_bwZwdmaQDZB77MfTf6pk_H67kv0N3qbvmsmqWIMVcGroyCnbzOUSdfSlhJMhDQXjfvaWFGHgP8HZXwZjKjoy3_QXZpzrJ2olGRKcCmXvy8PlM2plAzDKW-M9LIEelkC_XcJasTz2-We-D9Tr8CLI7BEnnSYVotmmJBKPDsQuzKlfMvAhCKip78B7LPELA</recordid><startdate>20160412</startdate><enddate>20160412</enddate><creator>van Ravesteyn, Thomas W.</creator><creator>Dekker, Marleen</creator><creator>Fish, Alexander</creator><creator>Sixma, Titia K.</creator><creator>Wolters, Astrid</creator><creator>Dekker, Rob J.</creator><creator>te Riele, Hein P. 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| 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 |
| title | LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells |
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