Directed Evolution of an Enhanced and Highly Efficient FokI Cleavage Domain for Zinc Finger Nucleases |
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Authors: | Jing Guo |
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Affiliation: | The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA |
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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 mutations that impart sequence specificity to the FCD that might be utilized in future studies to further refine ZFNs through cooperative specificity. In addition, Sharkey was observed to enhance the cleavage profiles of previously published and newly selected heterodimer ZFN architectures. This enhanced and highly efficient cleavage domain will aid in a variety of ZFN applications in medicine and biology. |
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Keywords: | ZFP, zinc finger protein ZFN, zinc finger nuclease DSBs, double-strand breaks NHEJ, nonhomologous end joining HR, homologous recombination OPEN, oligomerized pool engineering FCD, FokI cleavage domain SR, survival rate EGFP, enhanced green fluorescent protein HEK, human embryonic kidney CMV, cytomegalovirus |
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