Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases.

ABSTRACT: Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The high specificity and affinity of these chimeric enzymes are based on custom-designed zinc finger proteins (ZFPs). To improve the performance of existing ZFN technology, we developed an in vivo evolution-based approach to improve the efficacy of the FokI cleavage domain (FCD). After multiple rounds of cycling mutagenesis and DNA shuffling, a more efficient nuclease variant (Sharkey) was generated. In vivo analyses indicated that Sharkey is >15-fold more active than wild-type FCD on a diverse panel of cleavage sites. Further, a mammalian cell-based assay showed a three to sixfold improvement in targeted mutagenesis for ZFNs containing derivatives of the Sharkey cleavage domain. We also identified mu

SUBMITTER: Guo J 

PROVIDER: S-EPMC2885538 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature