An Improved Genome Engineering Method Using Surrogate Reporter-Coupled Suicidal ZFNs.

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.