Abstract
Using engineered nucleases such as zinc-finger nucleases (ZFNs) and TALE nuclease (TALEN) to accomplish genome editing often causes high cellular toxicity because of the consistent expression of artificial nucleases and off-targeting effect. And lacking selection marker in modified cells makes it hard to enrich these positive cells. Here we introduce a method by incorporating a surrogate reporter enrichment into a suicidal ZFN system, which is designed by a pair of ZFN expression cassettes flanked with its target sites. Our data demonstrated that this modified system achieved almost the same ZFN activity as the original method but reduced ~40% toxicity. This new suicidal ZFN expression system coupled with a surrogate reporter not only enables decreased cellular toxicity but also makes the genetic modified cells to be enriched by EGFP analysis.
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Acknowledgments
The authors thank the colleagues in Professor Zhang’s lab for their technical assistance and helpful collaboration. We are grateful to financial support from China’s Ministry of Agriculture (948 Program 2013-Z27), China’s Ministry of Science and Technology (National Science and Technology Major Project 2014ZX0801009B and 973 Program 2011CBA01002), and National Natural Science Foundation of China (NSFC 31172186).
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Xing, J. et al. (2018). An Improved Genome Engineering Method Using Surrogate Reporter-Coupled Suicidal ZFNs. In: Liu, J. (eds) Zinc Finger Proteins. Methods in Molecular Biology, vol 1867. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8799-3_13
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DOI: https://doi.org/10.1007/978-1-4939-8799-3_13
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8798-6
Online ISBN: 978-1-4939-8799-3
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