Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

Table 6

Evolutionary success of C₂H₂ binding proteins. Relevant observations concerning evolutionary success of C₂H₂ binding proteins.

Observations	References and comments
Degeneracy	(i) Engineered ZFAs typically yielded degenerate motifs, binding dozens to hundreds of related individual sequences [3].
	(ii) Observed clear secondary DNA binding preferences and the secondary motifs were bound nearly as well as the primary motifs [2].
	(iii) The secondary motif can recruit genomic loci independently of the primary motif [2].
	(iv) Beyond simply providing a DNA binding site motif, these data provide rank-ordered listing of the preference of a protein [2].
	(v) Observed “secondary motif” phenomenon had not been described before, and it has important implications for understanding how proteins interact with their DNA binding sites [2].
High failure rates	The modular assembly method of engineering zinc finger arrays has an unexpectedly higher failure rate [7].
Evolutionary plasticity	(i) The dramatic expansion of the number of C2H2-ZFs in mammals appears to be a recent evolutionary event [3].
	(ii) Evolutionary plasticity [34, 47].
	(iii) Conserved expression without conserved regulatory sequence: the more things change, the more they stay the same [1].
Complexity	(i) Half of the proteins: each recognized multiple distinctly different sequence motifs [2].
	(ii) 10605 combinations for a 1000 bp long gene [48].
	(iii) The dramatic expansion of the number of C2H2-ZFs in mammals appears to be a recent evolutionary event [3].
Simplicity	(i) Origami structure: [49–53].
	(ii) Fractal organization: [54–56].
Directional evolution	(i) Expression of ftz changed at least three times during arthropod evolution: [47].
	(ii) The complexity, robustness, and evolvability of regulatory systems [1].
Evolutionary traits	(i) “The contribution of finger 1 to the DNA binding affinity of SP1 is smaller than that of fingers 2 and 3, but the presence of finger 1 is still essential for the high DNA binding affinity. These unique features have never been detected in other zinc fingers [8].
Cytotoxicity	Cell death and apoptosis associated with ZFN expression are most likely the result of excessive cleavage at off-target sites, which, in turn, suggests imperfect target-site recognition by the ZF DNA-binding domains. [6, 39, 40]