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Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides
Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of biological systems. It is likely that gene editing is facilitated by the direct incorporation of the oligonucleotides via replication and/or by direct conversion, most lik...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113578/ https://www.ncbi.nlm.nih.gov/pubmed/21343181 http://dx.doi.org/10.1093/nar/gkr061 |
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author | Falgowski, Kerry Falgowski, Carly York-Vickers, Cassie Kmiec, Eric B. |
author_facet | Falgowski, Kerry Falgowski, Carly York-Vickers, Cassie Kmiec, Eric B. |
author_sort | Falgowski, Kerry |
collection | PubMed |
description | Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of biological systems. It is likely that gene editing is facilitated by the direct incorporation of the oligonucleotides via replication and/or by direct conversion, most likely through the DNA mismatch repair pathway. The phenomenon of strand bias, however, as well as its importance to the gene editing reaction itself, has yet to be elucidated in terms of mechanism. We have taken a reductionist approach by using a genetic readout in Eschericha coli and a plasmid-based selectable system to evaluate the influence of strand bias on the mechanism of gene editing. We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater ‘editing’ efficiency than ‘those that anneal to’ the leading strand. The majority of editing events (∼70%) occur by the incorporation of the ODN during replication within the lagging strand. Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway. In general, the influence of DNA replication is independent of which ODN is used suggesting that the importance of strand bias is a reflection of the underlying mechanism used to carry out gene editing. |
format | Online Article Text |
id | pubmed-3113578 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-31135782011-06-14 Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides Falgowski, Kerry Falgowski, Carly York-Vickers, Cassie Kmiec, Eric B. Nucleic Acids Res Molecular Biology Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of biological systems. It is likely that gene editing is facilitated by the direct incorporation of the oligonucleotides via replication and/or by direct conversion, most likely through the DNA mismatch repair pathway. The phenomenon of strand bias, however, as well as its importance to the gene editing reaction itself, has yet to be elucidated in terms of mechanism. We have taken a reductionist approach by using a genetic readout in Eschericha coli and a plasmid-based selectable system to evaluate the influence of strand bias on the mechanism of gene editing. We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater ‘editing’ efficiency than ‘those that anneal to’ the leading strand. The majority of editing events (∼70%) occur by the incorporation of the ODN during replication within the lagging strand. Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway. In general, the influence of DNA replication is independent of which ODN is used suggesting that the importance of strand bias is a reflection of the underlying mechanism used to carry out gene editing. Oxford University Press 2011-06 2011-02-22 /pmc/articles/PMC3113578/ /pubmed/21343181 http://dx.doi.org/10.1093/nar/gkr061 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Molecular Biology Falgowski, Kerry Falgowski, Carly York-Vickers, Cassie Kmiec, Eric B. Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides |
title | Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides |
title_full | Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides |
title_fullStr | Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides |
title_full_unstemmed | Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides |
title_short | Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides |
title_sort | strand bias influences the mechanism of gene editing directed by single-stranded dna oligonucleotides |
topic | Molecular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113578/ https://www.ncbi.nlm.nih.gov/pubmed/21343181 http://dx.doi.org/10.1093/nar/gkr061 |
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