Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?  Annett Strauβ, Thomas Lahaye  Molecular Plant  Volume 6, Issue 5, Pages 1384-1387 (September 2013) DOI: 10.1093/mp/sst075 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Programmable DNA Binding Domain Architectures and Derived Designer Nucleases. Zinc finger nucleases (ZFNs) are typically composed of three or four tandem arranged fingers (four-finger ZF is displayed), each binding three base pairs and translationally fused to a FokI nuclease domain. Greatest success has been achieved with zinc fingers consisting of GNN triplets. Interaction of two distinct ZF arrays with their DNA target results in dimerization and activation of FokI. Thus, both ZF arrays contribute additively to target specificity. TAL effector nucleases (TALENs) also typically rely on a fused FokI domain. The TALE DNA binding domain is composed of tandem arranged repeats, each binding one base. The number of repeats is generally highly flexible but typically custom arrays contain about 16–24 repeats (17.5 repeat array is displayed). Recent studies show that ‘unfavored’ base–residue pairings in the N-terminus of the repeat array (5’ of target DNA) have a stronger impact on the interaction with DNA than mispairing in the C-terminus (black triangle; Meckler et al., 2013). RNA-guided endonucleases (RGENs) consist of a protein (Cas9) scaffold with two cleavage domains (RuvC and HNH) and a custom guide RNA (gRNA) that defines specificity. A conserved protospacer adjacent motif (PAM) that limits target flexibility is indicated. Mismatches in the 3’ end of the protospacer target DNA (3’ of guide RNA) have stronger impact on the cleavage activity of RGENs than mismatches in the 5’ end (black triangle; Jinek et al., 2012; Cong et al., 2013; Jiang et al., 2013). Green elements indicate protein or RNA elements that mediate DNA binding. Red and green font indicate invariant and programmable bases, respectively. Numbers above and between the double arrows indicate numbers of bases of the target region and the optimal spacing, respectively. The number of bases that can be programmed without restrictions is given in brackets. Please note that the size of the targeted region and the ideal spacing is to some extent variable and dictated by the given TALEN or ZFN architecture. Molecular Plant 2013 6, 1384-1387DOI: (10.1093/mp/sst075) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions