Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Falgowski, Kerry, Falgowski, Carly, York-Vickers, Cassie, Kmiec, Eric B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2011
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
_version_ 1782205947796520960
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
work_keys_str_mv AT falgowskikerry strandbiasinfluencesthemechanismofgeneeditingdirectedbysinglestrandeddnaoligonucleotides
AT falgowskicarly strandbiasinfluencesthemechanismofgeneeditingdirectedbysinglestrandeddnaoligonucleotides
AT yorkvickerscassie strandbiasinfluencesthemechanismofgeneeditingdirectedbysinglestrandeddnaoligonucleotides
AT kmiecericb strandbiasinfluencesthemechanismofgeneeditingdirectedbysinglestrandeddnaoligonucleotides