Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications.
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.
Radecke
Sarah
S
Department of Molecular Diagnostics, Molecular Therapy and Experimental Transplantation, Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Donation Service Baden-Wuerttemberg-Hessen, Ulm, Germany.
Radecke
Frank
F
Cathomen
Toni
T
Schwarz
Klaus
K
D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D052416:Targeted Gene Repair / Q000379:methods*; D016335:Zinc Fingers
D009838:Oligodeoxyribonucleotides; D003850:Deoxyribonuclease I