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). In order 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 3 to 6-fold 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.

Precision genome engineering with programmable DNA-nicking enzymes

“…We propose that the efficiency of nickase-mediated genome editing might be enhanced by altering culture conditions (Doyon et al 2010), using a reporter-based enrichment method (Kim et al 2011a), or improving the FokI domain via directed evolution (Guo et al 2010;Doyon et al 2011), all of which have been useful for enhancing the activity of nucleases. FokI is an enzyme that is evolutionarily programmed to produce DSBs but not SSBs.…”

TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy

“…Together, the results from our study suggest that TALENs have potential as a therapeutic strategy for HPV infection and related cervical malignancy. cated by SSA reporter assay ( Figure 1E), FokIs, which carried ELD/ KKR (15) and Sharkey mutations (16), generally demonstrated better efficiency than the other variants; when fused with the C-terminal +63 truncation architecture, FokI induced the highest frequency of DNA breaks with comparably low toxicity ( Figure 1F). The efficiencies of these combinations were further confirmed using a surrogate reporter system (Supplemental Figure 2 and ref.…”

Highly active zinc-finger nucleases by extended modular assembly

“…As a more pragmatic solution, longer arrays of modules could be used to increase affinity. Anecdotal evidence supported this concept (Gordley et al 2009;Guo et al 2010;Perez-Pinera et al 2012). However, we showed recently that a caveat to the latter approach is that the addition of modules can, in some cases, reduce the activity of a ZFN, perhaps by allowing subgroups of fingers to bind additional unexpected locations (Shimizu et al 2011).…”