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Evaluating the mutagenic activity of targeted endonucleases containing a Sharkey FokI cleavage domain variant in zebrafish.

Zebrafish (2013-06-21)
Laura M Pillay, Lyndsay G Selland, Valerie C Fleisch, Patricia L A Leighton, Caroline S Cheng, Jakub K Famulski, R Gary Ritzel, Lindsey D March, Hao Wang, W Ted Allison, Andrew J Waskiewicz
ABSTRACT

Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Nde I from Neisseria denitrificans, Restriction Enzyme