Expanding or Restricting the Target Site Repertoire of Zinc-finger Nucleases: The Inter-domain Linker as a Major Determinant of Target Site Selectivity

Charité Medical School, Institute of Virology (CBF), Berlin, Germany.
Molecular Therapy (Impact Factor: 6.23). 12/2008; 17(1):104-11. DOI: 10.1038/mt.2008.233
Source: PubMed


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.

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Available from: Toni Cathomen
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    • "This stereotyped binding mode has made ZFs an object of intense research for the design of artificial TFs and custom ZF nucleases for site-specific genome editing (Klug, 2010). Despite the appeal of a simple recognition code based on a few canonical residues, additional features of ZF proteins can affect DNA-binding specificity, including interdomain interactions (Isalan et al., 1997; Liu and Stormo, 2008; Wolfe et al., 1999), the interdomain linker sequence (Handel et al., 2009), ZF docking geometry (Siggers and Honig, 2007), and residues outside the canonical recognition residues (Persikov and Singh, 2011). Residues in the loop between the b strands (i.e., b turn) can also affect DNA binding affinity and footprinting pattern (Shiraishi et al., 2005). "
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    ABSTRACT: A major challenge in obtaining a full molecular description of evolutionary adaptation is to characterize how transcription factor (TF) DNA-binding specificity can change. To identify mechanisms of TF diversification, we performed detailed comparisons of yeast C2H2 ZF proteins with identical canonical recognition residues that are expected to bind the same DNA sequences. Unexpectedly, we found that ZF proteins can adapt to recognize new binding sites in a modular fashion whereby binding to common core sites remains unaffected. We identified two distinct mechanisms, conserved across multiple Ascomycota species, by which this molecular adaptation occurred. Our results suggest a route for TF evolution that alleviates negative pleiotropic effects by modularly gaining new binding sites. These findings expand our current understanding of ZF DNA binding and provide evidence for paralogous ZFs utilizing alternate modes of DNA binding to recognize unique sets of noncanonical binding sites.
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    • "In brief, all nucleases contained an amino-terminal hemagglutinin tag and an SV40 nuclear localization signal. The codon-optimized, AAVS1-specific zincfinger arrays were synthesized by GeneArt/Life Technologies and fused to the obligate heterodimeric FokI variant EA-KV (Szczepek et al., 2007) by the 4 AA linker LRGS (Händel et al., 2009). TALENs are based on the ND134/C+17 architecture (Mussolino et al., 2011) and contain wild-type FokI nuclease domains. "

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    • "First, nucleases were targeted to a site in the first exon of each gene in order to increase the likelihood that a frame shift would terminate translation upstream of known functional domains, particularly the homeodomain. Second, the spacing between the target sequences for each ZFN pair was set to either 5 bp or 6 bp based on previous reports indicating that these represent optimal spacing [34]. Third, we targeted regions containing a restriction site that could be used to screen for mutations. "
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