Quantification of zinc finger nuclease-associated toxicity.
The recent development of artificial zinc finger nucleases (ZFNs) for targeted genome editing has opened a broad range of possibilities in biotechnology and gene therapy. The ZFN technology allows a researcher to deliberately choose a target site in a complex genome and create appropriate nucleases to insert a DNA double-strand break (DSB) at that site. Gene editing frequencies of up to 50% in non-selected human cells attest to the power of this technology. Potential side effects of applying ZFNs include toxicity due to cleavage at off-target sites. This can be brought about by insufficient specificity of DNA binding, hence allowing ZFN activity at similar target sequences within the genome, or by activation of the ZFN nuclease domains before the nuclease is properly bound to the DNA. Here, we describe two different methods to quantify ZFN-associated toxicity: the genotoxicity assay is based on quantification of DSB repair foci induced by ZFNs whereas the cytotoxicity is based on assessing cell survival after application of ZFNs.
Cornu
Tatjana I
TI
Institute of Virology (CBF), Charité Medical School, Berlin, Germany.
Cathomen
Toni
T
D002460:Cell Line; D002470:Cell Survival / Q000235:genetics / Q000502:physiology*; D053903:DNA Breaks, Double-Stranded; D004720:Endonucleases / Q000235:genetics / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D016335:Zinc Fingers / Q000235:genetics*
D004720:Endonucleases