DNA Breakage Associated with Targeted Gene Alteration Directed by DNA Oligonucleotides

Overview

Journal Mutat Res

Publisher Elsevier

Specialty Genetics

Date 2009 May 26

PMID 19463835

Citations 18

Authors

Melissa Bonner

Eric B Kmiec

Affiliations

Soon will be listed here.

Abstract

Understanding the mechanism by which single-stranded oligonucleotides (ODNs) elicit targeted nucleotide exchange (TNE) is imperative to achieving optimal correction efficiencies and medical applicability. It has been previously shown that introduction of an ODN into cells results in the activation of DNA damage response pathways, but there has been no evaluation of the damage created at the level of the DNA. The activation of H2AX, a hallmark protein of DNA breakage, suggests that a double-strand break (DSB) could be occurring during the targeted gene alteration (TGA) reaction. Using the human HCT116 cell line with a single integrated mutant eGFP gene as our model system, we demonstrate that the DNA strand breakage occurs when a specific ODN, designed to direct TGA, is transfected into the cells. Both single- and double-stranded DNA cleavage is observed dependent on the level of ODN added to the reaction. Possible mechanisms of ODN-dependent DSB formation, as a function of TGA, are discussed herein.

Citing Articles

Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells.

Tiricz H, Nagy B, Ferenc G, Torok K, Nagy I, Dudits D J Plant Res. 2017; 131(1):179-189.

PMID: 28836127 DOI: 10.1007/s10265-017-0975-8.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

Rivera-Torres N, Banas K, Bialk P, Bloh K, Kmiec E PLoS One. 2017; 12(1):e0169350.

PMID: 28052104 PMC: 5214427. DOI: 10.1371/journal.pone.0169350.

Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells.

Santos D, Kiskinis E, Eggan K, Merkle F Curr Protoc Stem Cell Biol. 2016; 38:5B.6.1-5B.6.60.

PMID: 27532820 PMC: 4988528. DOI: 10.1002/cpsc.15.

λ Recombination and Recombineering.

Murphy K EcoSal Plus. 2016; 7(1).

PMID: 27223821 PMC: 11575712. DOI: 10.1128/ecosalplus.ESP-0011-2015.

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

Bialk P, Rivera-Torres N, Strouse B, Kmiec E PLoS One. 2015; 10(6):e0129308.

PMID: 26053390 PMC: 4459703. DOI: 10.1371/journal.pone.0129308.

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