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 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.

Figure 1. Schematic representation of the selection strategy used for isolating novel FCD variants

(a,b) A two-plasmid approach utilizing a reporter consisting of a single ZFN cleavage site downstream of ccdB and a ZFN expression plasmid under tight control of a modified lac promoter can be used to selectively enrich for catalytically improved FokI cleavage domains. (c) A library of FCD variants can be transformed into the ccdB harboring BW25141 selection strain and enriched following ZFN mediated reporter plasmid cleavage/degradation. Decreasing the recovery time following transformation facilitates the isolation of ZFN variants with enhanced catalytic properties.

Figure 2. Enhancing the FokI cleavage domain by directed evolution

A library of ZFNs was transformed into selection strain BW25141 and subjected to multiple rounds of evolution. (a) Survival rate (SR), which correlates directly with catalytic activity, was measured at 1 hr for Rounds 3, 6 and 9. (b) SR was observed to increase with recovery time. SR curves were measured for wt (◆), R3 (■), R6 (▲) and R9 (●). (c) The extent of substrate linearization from Rounds 3, 6 and 9 was measured from cellular extracts prepared from overnight cultures. ‘Sub’ indicates supercoiled substrate plasmid pSub-P3. ‘Prod’ indicates linearized substrate plasmid pSub-P3. (d) SR for selection rounds 11–18 measured at 1 hr. Bar 10 indicates FCD_R18-28.

Figure 3. Selected ZFN variants have enhanced catalytic profiles as demonstrated by in vitro DNA cleavage assays and in vivo activity assays

(a) In vitro cleavage of target DNA by P3.nuclease with either wild-type FokI cleavage domain (white) or FCD_R18-28 (black). Cleavage rates were determined by measuring the initial velocity of DNA cleavage. 12 nM ZFN was added to pre-warmed ZFN Reaction Buffer containing 4, 6, 12, 24, or 36 nM pSub-P3 substrate DNA. Reaction conditions were standardized. (b) In vitro cleavage of target DNA by P3 nuclease with either FCD_R18-28 or wild-type FokI cleavage domain. ‘Uncut’ indicates linearized substrate plasmid pSub-P3. ‘Cut’ indicates cleavage products. Cleavage was monitored incrementally over 90 min. (c) Activity analysis of FokI cleavage domain variants containing the selected mutations S418P, K441E, Q481H, N527D and S418P::K441E with the P3 zinc finger domain. ZFN activity was measured against MluI (ACGGCT) and Nx6 (N: A, T, C or G) spacer sequences and normalized to wild-type FCD. Error bars indicate standard deviation of three replicates. (d) Activity analysis of FokI cleavage domain variants S418P::K441E (Sharkey) and FCD_R18-28 (Sharkey’) with the P3 zinc finger domain. ZFN activity was measured against MluI, Nx6, ACGAAT, VF2471 (GAGAGT) and CFTR (TGGTGA) spacer sequences and normalized to wild-type FCD. Error bars indicate standard deviation of three replicates.

Figure 4. Sharkey’ increases the rate of mutagenesis in a mammalian model system

(a) Schematic overview of the reporter system used to evaluate the efficiency of mutagenesis in mammalian cells. The model system consists of a HEK 293 cell line containing a modified and disabled EGFP transgene stably integrated in a single locus. An MluI restriction site flanked by E2C zinc finger recognition sites was inserted between EGFP residues 157 and 158. Select deletions (e.g., 2, 5 or 8-bp) or insertions (e.g., 1, 4, or 7-bp) result in frame restoration and EGFP expression. (b) Representative flow cytometry data for reporter cells transfected with CMV controlled wild-type FokI and Sharkey’ cleavage domains with 3, 4, 5 and 6-finger zinc finger DNA binding domains. Mutagenesis is measured by counting the % of EGFP positive cells. (c) Quantification of EGFP positive reporter cells following transfection with ZFN. Error bars denote standard deviation of three replicates (d) MluI restriction digest assay of HEK 293 reporter cells transfected with ZFN. ‘Cut’ indicates the presence of unmodified reporter gene. ‘Uncut’ indicates the presence of ZFN modified reporter gene. The % of modified reporter cells is indicated.

Figure 5. Sharkey is compatible with alternative ZFN architectures

Mutagenesis efficiencies for asymmetric ZFN scaffolds comprised of wild-type FokI cleavage domain (black) or Sharkey mutations (white). (a) RR::DD, RR::DS and (b) KK::EL FCD architectures were fused to E4 or P3.ZFPs. Stimulated mutagenesis was measured by the counting % of EGFP positive cells. Error bars indicate standard deviation of three replicates.

Figure 6. Selected mutations mapped on the crystal structure of the restriction endonuclease FokI

Mutations identified in ≥70% sequenced variants are highlighted. Activating mutations S418P and K441E are depicted as red spheres on full-length FokI bound to substrate DNA (PDB ID: 1FOK) . The selected mutations Q481H and N527D are shown as blue spheres. The catalytic center amino acids Asp 450, Asp 467 and Lys 469 are depicted as green balls and sticks.