Cellular responses to targeted genomic sequence modification using single-stranded oligonucleotides and zinc-finger nucleases

Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

“…S5 in the supplemental material). These results suggest that hairpin formation that is detectable by ZFN digestion does not activate the intra-S-phase checkpoint and that in contrast to chemically modified ODNs (23,24,27), ODN-mediated dissolution of replication fork hairpins does not provoke RPA-dependent checkpoint activation. The absence of Chk1 phosphorylation during drug treatment or ODN administration also argues that sufficient ssDNA is not generated at the replication fork to activate the ATR-Chk1 DNA damage response, nor do the 5= and 3= termini of the annealed ODNs lead to checkpoint activation.…”

“…Compared to phosphorothioate-protected or locked nucleic acids, unmodified ODNs minimally reduce the viability of targeted cells or induce DNA damage response checkpoints in mouse embryonic stem cells (23,27). Based on this consideration, we introduced unmodified ODNs into HeLa/c-myc-(CTG) n · (CAG) n cell lines, in which replication fork movement from the adjacent c-myc replicator unambiguously defines the leading and lagging template strands.…”

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.…”

“…59 In addition, inducible repression of hMLH1 resulted in a threefold increase in targeting frequency in a doxycycline-regulatable HEK293T La cell line. 6 Although MMR downregulation consistently resulted in increased targeting frequencies, the extent of the increase varied between the different studies. Evidently, this variation could be explained by differences in the degree of knockdown that had been achieved.…”

“…Homology-directed repair of the double-stranded DNA break using a linear double-stranded donor DNA fragment carrying the desired alteration may be accompanied by site-specific alteration of the genome. 5,6 Although this approach can be remarkably effective (single base changes have been obtained in±20% of exposed cells), an obvious obstacle is that zinc-finger nucleases need to be designed and constructed for every specific location to be modified. Zinc-finger nucleases-directed gene modification may therefore be particularly useful to generate a series of substitutions at the same location or, with a view on gene therapy, to repair a frequently occurring mutation.…”