Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity

Abstract

Precise manipulations of complex genomes by zinc-finger nucleases (ZFNs) depend on site-specific DNA cleavage, which requires two ZFN subunits to bind to two target half-sites separated by a spacer of 6 base pairs (bp). ZFN subunits consist of a specific DNA-binding domain and a nonspecific cleavage domain, connected by a short inter-domain linker. In this study, we conducted a systematic analysis of 11 candidate-based linkers using episomal and chromosomal targets in two human cell lines. We achieved gene targeting in up to 20% of transfected cells and identified linker variants that enforce DNA cleavage at narrowly defined spacer lengths and linkers that expand the repertoire of potential target sites. For instance, a nine amino acid (aa) linker induced efficient gene conversion at chromosomal sites with 7- or 16-bp spacers, whereas 4-aa linkers had activity optima at 5- and 6-bp spacers. Notably, single aa substitutions in the 4-aa linker affected the ZFN activity significantly, and both gene conversion and ZFN-associated toxicity depended on the linker/spacer combination and the cell type. In summary, both sequence and length of the inter-domain linker determine ZFN activity and target-site specificity, and are therefore important parameters to account for when designing ZFNs for genome editing.

Figure 1

Zinc-finger nuclease (ZFN) linker variants. (a) Schematic of the EB2-N homodimer. All ZFN variants are based on EB2-N²³ and contain an N-terminal HA epitope tag, three zinc-fingers (1, 2, 3), the inter-domain linker, and the FokI cleavage domain. The two target half-sites (L and R) are separated by a spacer sequence of defined length (see Supplementary Table S1). (b) Sequence of ZFN linker variants. The names, linker lengths, and the relevant amino acid sequences are indicated. The ZFN inter-domain linker length has been defined as the number of amino acids (aa) between the last conserved histidine in the third zinc-finger (F3) and the first residue of the FokI cleavage domain (QLV). Note that in some variants the first residues of the FokI domain were deleted and offset against the linker length. (c) Expression analysis of ZFN linker variants. HEK293T cells were transfected with an expression plasmid encoding a ZFN linker variant along with pEGFP as an internal transfection control. After 30 h, the cells were harvested and the lysates were probed simultaneously with antibodies against both the HA tag and EGFP. HA-tagged I-SceI and mock-transfected cells (cto) were included as controls. The positions of the various ZFNs, I-SceI, EGFP, and size markers are indicated.

Figure 2

Activity profile of the zinc-finger nuclease (ZFN) linker variants on episomal targets. (a) Experimental setup. The donor and target plasmids are described in the text. The positions of the two binding half-sites for the homodimeric ZFN linker variants (gray boxes), the spacer sequence (white box), and the ‐1 frameshift are indicated in the target. TGA designates the stop codon of the lacZ gene while GTG represents the first triplet of the EGFP open reading frame. Arrows denote the positions of the primers used for genotyping. (b) Episomal recombination assay. HEK293T cells were transfected with plasmids encoding donor, target, and ZFN expression vectors. The columns designate the percentage of EGFP-positive cells 2 days after transfection, as determined by flow cytometry. The respective linker variants and spacer lengths are indicated. In cto, a target containing a recognition site for I-SceI but not for the ZFNs was used. For all other targets, I-SceI was included as a negative control to indicate the level of nonstimulated gene targeting. Note the change in scale between the first and second graph and that column labels alternate. (c) Activity profile of the ZFN linker variants. Data from b was replotted for all functional ZFN linker variants and itemized according to linker length to display target site selectivity. A statistically significant increase in gene targeting relative to nonstimulated HR is indicated by * (P < 0.05) or ** (P < 0.01).

Figure 3

Influence of spacer sequence on zinc-finger nuclease (ZFN) activity. HEK293T cells were transfected with plasmids encoding ZFN expression vectors, donor, and targets containing an AT-rich spacer, a GC-rich spacer, or a balanced 6-bp spacer. The bars designate the percentage of EGFP-positive cells 2 days after transfection as determined by flow cytometry. The respective linker variants and the sequence of the spacers are indicated. I-SceI was included as a negative control to indicate the level of nonstimulated gene targeting. A statistically significant increase in gene targeting relative to nonstimulated HR is indicated by * (P < 0.05) or ** (P < 0.01).

Figure 4

Activity profile of the zinc-finger nuclease (ZFN) linker variants on chromosomal targets. (a) Chromosomal recombination assay. HEK293-based cell lines that contain an integrated version of the target loci used in Figure 2 were transfected with the various ZFN expression vectors, a donor plasmid, and a DsRed-Express (REx) expression vector to normalize for transfection efficiency. The columns designate the percentage of EGFP-positive cells 5 days after transfection as determined by flow cytometry. The respective linker variants and spacer lengths are indicated. In cto, a cell line containing an integrated target locus with a recognition site for I-SceI but not for the ZFNs was used. For all other target cell lines, I-SceI was included as a negative control to indicate the level of nonstimulated gene targeting. (b) Activity profile of the ZFN linker variants. Data from a was replotted for all functional ZFN linker variants and itemized according to linker length to display target site selectivity of the linker variants. A statistically significant increase in gene targeting relative to nonstimulated HR is indicated by * (P < 0.05) or ** (P < 0.01).

Figure 5

Cell line dependence of zinc-finger nuclease (ZFN)-mediated gene targeting and ZFN-associated toxicity. (a) Chromosomal recombination assay. U2OS/6 (black bars) and 293/6 (gray bars) target cell lines containing an integrated lacZfsEGFP locus with a 6-bp spacer were transfected with the various ZFN expression vectors, a donor plasmid, and a DsRed-Express (REx) expression vector. The REx marker was used to normalize for transfection efficiency and to follow the fate of transfected cells over time. The columns designate the percentage of EGFP-positive cells 5 days after transfection as determined by flow cytometry. I-SceI was included as a negative control to indicate the level of nonstimulated gene targeting. A statistically significant increase in gene targeting relative to nonstimulated HR is indicated by * (P < 0.05) or ** (P < 0.01). (b) Genotyping. U2OS/6 target cells were transfected as described in a and genomic DNA was isolated 5 days later. A nested PCR was performed with primers designed to amplify the corrected target locus (cTL). The positions of the primers used for the nested PCR protocol are indicated in Figure 2a. As an internal quality control, a fragment of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) locus was amplified. (c) Cell survival assay. U2OS/6 (black bars) and 293/6 (gray bars) target cells were transfected as described in a and the percentage of REx-positive cells was determined by flow cytometry 2 and 5 days after transfection. The columns represent the fraction of REx-positive cells at day 5 as compared to the fraction at day 2 after transfection, and are shown relative to transfection with an I-SceI expression plasmid. A statistically significant decrease in survival as compared to I-SceI is indicated by * (P < 0.05) or ** (P < 0.01).