Zinc-finger Nuclease-induced Gene Repair With Oligodeoxynucleotides: Wanted and Unwanted Target Locus Modifications

Abstract

Correcting a mutated gene directly at its endogenous locus represents an alternative to gene therapy protocols based on viral vectors with their risk of insertional mutagenesis. When solely a single-stranded oligodeoxynucleotide (ssODN) is used as a repair matrix, the efficiency of the targeted gene correction is low. However, as shown with the homing endonuclease I-SceI, ssODN-mediated gene correction can be enhanced by concomitantly inducing a DNA double-strand break (DSB) close to the mutation. Because I-SceI is hardly adjustable to cut at any desired position in the human genome, here, customizable zinc-finger nucleases (ZFNs) were used to stimulate ssODN-mediated repair of a mutated single-copy reporter locus stably integrated into human embryonic kidney-293 cells. The ZFNs induced faithful gene repair at a frequency of 0.16%. Six times more often, ZFN-induced DSBs were found to be modified by unfaithful addition of ssODN between the termini and about 60 times more often by nonhomologous end joining-related deletions and insertions. Additionally, ZFN off-target activity based on binding mismatch sites at the locus of interest was detected in in vitro cleavage assays and also in chromosomal DNA isolated from treated cells. Therefore, the specificity of ZFN-induced ssODN-mediated gene repair needs to be improved, especially regarding clinical applications.

Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases

“…If the break is repaired by NHEJ, which is an error-prone repair mechanism, such repaired target sequences frequently harbor insertions or deletions (Bibikova et al, 2002;Cong et al, 2013;Mali et al, 2013;Miller et al, 2011). Alternatively, it is possible to edit target sequences precisely through a DSB-induced HDR mechanism by introducing into cells both nucleases and a DNA template, such as singlestranded oligonucleotides (Bedell et al, 2012;Cong et al, 2013;Radecke et al, 2010;Wang et al, 2013) or longer dsDNA donors (Cong et al, 2013;Hockemeyer et al, 2011;Urnov et al, 2005). Therefore, custom nucleases can theoretically be employed to modify genomes of any genetic model organism.…”

Electrospun fiber membranes enable proliferation of genetically modified cells

“…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.…”

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

“…An advanced OTNE technology can be a key component in the combinatorial use of oligonucleotides with programmable nucleases. In human and mouse cells the expression of either ZFNs or TALENs in combination with SDO stimulated OTNE-mediated gene repair (Radecke et al 2010;Strouse et al 2014;Wefers et al 2013). Recent publications provide further examples for plant gene editing by co-transformation of SDO molecules and sequence-specific nucleases (Svitashev et al 2015;Wang et al 2015).…”