Abstract
We describe a method for detecting and validating genomic aberrations arising from cell lines exposed to zinc finger nucleases (ZFNs), an important reagent used for targeted genome modifications. This method makes use of cloned cell lines, an approach that adds power when testing variables that may affect gene correction efficiency and evaluating potential side effects on a genome-wide scale. After cell treatment, the genomic DNA isolation method, as described, is ideal for high-resolution array comparative genomic hybridization (aCGH) and quantitative PCR. Guidelines for aCGH analysis and calling significant copy number variations (CNVs) for validation by qPCR are also discussed. Using this method, we describe a novel ZFN-associated chromosome 4 copy number variation (CNV) attributable to a predicted ZFN off-target cleavage site found within the CNV.
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Acknowledgement
The authors would like to thank Matthew Porteus, University of Stanford, for supplying GFP-293T cells and GFP-ZFN plasmids SP202A, SP202B and PC264 donor substrate. We also thank Susanna Lewis for comments and revisions to the chapter. This work was supported by the NIH Nanomedicine Center for Nucleoprotein Machines PN2 EY018244.
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Lindsay, C.R., Roth, D.B. (2014). An Unbiased Method for Detection of Genome-Wide Off-Target Effects in Cell Lines Treated with Zinc Finger Nucleases. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_23
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DOI: https://doi.org/10.1007/978-1-62703-761-7_23
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-761-7
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