Targeted manipulation of mammalian genomes using designed zinc finger nucleases

Abstract

Targeted introduction of a double-stranded break (DSB) using designer zinc finger nucleases (ZFNs) in mammalian cells greatly enhances gene targeting - homologous recombination (HR) at a chosen endogenous target gene, which otherwise is limited by low spontaneous rate of HR. Here, we report that efficient ZFN-mediated gene correction occurs at a transduced, transcriptionally active, mutant GFP locus by homology-directed repair, and that efficient mutagenesis by non-homologous end joining (NHEJ) occurs at the endogenous, transcriptionally silent, CCR5 locus in HEK293 Flp-In cells, using designed 3- and 4-finger ZFNs. No mutagenesis by NHEJ was observed at the CCR2 locus, which has ZFN sites that are distantly related to the targeted CCR5 sites. We also observed efficient ZFN-mediated correction of a point mutation at the endogenous mutant tyrosinase chromosomal locus in albino mouse melanocytes, using designed 3-finger ZFNs. Furthermore, re-engineered obligate heterodimer FokI nuclease domain variants appear to completely eliminate or greatly reduce the toxicity of ZFNs to mammalian cells, including human cells.

Figure 1

ZFN-mediated gene targeting in HEK293 cells. (A) Schematic representation of ZFN-mediated gene correction of the mutant eGFP locus (disrupted with either the hCCR5 or mTYR ZFN target sites) in HEK293 Flp-In cells. ZFN-mediated gene correction of the mutant eGFP locus using hCCR5-OHD3-FN, 3-finger hCCR5-specific ZFNs containing the FokI nuclease domain variants reported by Miller et al. (19) (B) and mTYR-OHD1-FN, 3-finger mTYR-specific ZFNs containing the FokI nuclease domain variants generated at PBPL (C). GFP positive cells were seen 5 days post-transfection of HEK293 cells with 3-finger ZFNs and donor plasmid. No GFP positive cells were seen by microscopy after transfection of HEK293 cells with donor plasmid alone. OHD depicts obligate heterodimers.

Figure 2

Efficiency and efficacy of ZFN-mediated gene correction in HEK293 cells using 4-finger CCR5-specific ZFNs fused to different FokI nuclease domain variants. Panel (A) brightfield; Panel (B) GFP positive cells seen post-transfection of HEK293 Flp-In cells with 4-finger CCR5-ZFNs and donor plasmids; Panel (C) FACS analyses (Q4 shows GFP⁺Cells). WT-FN, CCR5-ZFN constructs carrying the wild-type FokI nuclease domains. OHD depicts obligate heterodimers. OHD1-FN, CCR5-ZFN constructs carrying the FokI nuclease domain mutants that were generated at PBPL. OHD2-FN, CCR5-ZFN constructs carrying the FokI nuclease domain mutants reported by Szczepek et al., 2007 (20). OHD3-FN, CCR5-ZFN constructs carrying the FokI nuclease domain mutants reported by Miller et al., 2007 (19). (D) Frequency of gene correction in HEK293 Flp-In cells of a chromosomal mutant GFP reporter disabled by insertion of the CCR5 ZFN target sequence. Quantitative FACS analyses of the GFP positive cells at 3, 5 and 7 days post-transfection with designer ZFNs and donor plasmids. WT-FN, ZFN constructs carrying the wild-type FokI nuclease domains. (E) Analysis of the genotype of four different individual GFP positive clones. Five days post-transfection with ZFNs and the donor plasmids, GFP positive cells were sorted, serially diluted to get individual clones and grown. The genomic DNA was isolated from the GFP positive clones and the eGFP gene at the Flp-In locus was PCR amplified and digested with BstXI. The mutant eGFP gene has two BstXI sites, where the ZFN binding sites are inserted. Correction of the eGFP gene by homology-directed repair results in the loss of the BstXI sites. The PCR product size of the corrected eGFP gene is 930 bp as compared to 990 bp for the mutant gene. BstXI digestion of the mutant eGFP PCR product generates two bands: 450 bp and 540 bp, respectively. Lanes: Control, PCR product of the mutant eGFP gene from untransfected cells before (-) and after (+) digestion with BstXI; GFP⁺1-4, PCR products of 4 different individual clones obtained from GFP positive sorted cells before (-) and after (+) digestion with BstXI; M, 1 Kb ladder. All GFP positive cells are resistant to BstXI digestion, confirming ZFN-mediated eGFP gene correction in these cells.

Figure 3

Phenotypic and genotypic changes in the albino mouse melanocytes. A) Four different tyrosinase gene-corrected pigmented melanocytes 6 days post nucleofection of Melan-C cells (albino melanocytes) with the TYR ZFNs and the correcting plasmid (Tyrs J), are shown. B) Three pigmented clones (that had originated from individual pigmented cells) were obtained and correction of the mTYR gene was monitored by RFLP analysis (18). Presence of both 179 bp (a marker for the wild-type) and 144 bp (a marker for the mutant allele) fragments suggest targeted correction of one of the mutant tyrosinase alleles in the pigmented mouse albino melanocytes.