DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides

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.

Electrospun fiber membranes enable proliferation of genetically modified cells

“…[35][36][37] Briefly, cells were synchronized with 6 µM aphidicolin followed by 4 hours of release prior to introduction of the 3′ PTO (phosphorothioate)-modified 72-nucleotide (NT) singlestranded oligodeoxynucleotide (ssODN) by electroporation. One million cells in 100 µL of Hyclone McCoy's 5A serum free medium (Thermo Scientific, Logan, UT, USA) were mixed with the ssODN (4 µM) in an electroporation cuvette with a 4 mm gap (Fisher Scientific, Hampton, NH, USA).…”

“…The active components of these techniques, oligonucleotides, zinc finger nucleases, and transcription activator-like effector nucleases, [30][31][32] must be delivered into the target cell at levels that can have unintended effects -for example, zinc finger nuclease-offsite targeting. 33,34 One documented side effect is the phenomenon known as "reduced proliferation phenotype," 35 in which cells that have undergone genetic modification have inherently reduced rates of replication and proliferation. 36 This barrier needs to be removed before clinical applications of nanomedicine are designed and realized.…”

“…We have previously employed this type of system to evaluate gene editing in HCT116 cells. 30,35,36 The method involves synchronizing HCT116-19 cells using aphidicolin for 24 hours followed by a 4-hour release period prior to introduction of 72 NT DNA oligonucleotides by electroporation ( Figure 5A). Immediately after electroporation, 0.5 × 10 6 cells are plated onto a polylysinecoated dish or an electrospun fiber membrane for recovery and expansion.…”

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

“…This G 2 arrest was due to the presence of unrepaired genomic double-stranded DNA breaks, leading to the activation of the ATM/ATR pathway and phosphorylation of histone H2AX. 6,62,63 Although the mechanism underlying this ssODN-mediated toxicity is still largely unknown, the observed DNA damage response seemed to be induced particularly by ssODNs containing protective PTO linkages. 19 Several attempts have been made to counteract the apparent loss of targeted cells due to DNA damage caused by chemically-modified ssODNs.…”

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

“…Our current knowledge on the basic mechanisms of OTNE originates from several studies performed exclusively on microorganisms and mammalian cells (Bonner and Kmiec 2009;Engstrom and Kmiec 2007;Sargent et al 2011). According to the widely accepted general model, after invading the targeted site of the duplex DNA, SDOs are hybridized to the complementary strand through transient D-loop formation.…”