3.6 Zinc Finger Fusions to Transposases and ecombinases 41The Surveyor nuclease can be used to evaluate if the desired modification was
achieved, but it cannot be used to evaluate the number of off-target events in an
unbiased manner. The specificity of genome modification is also highly impor-
tant, whether one is selecting a gene knockout by limiting dilution or determin-
ing the percentage of off-target modifications in a pool to make inferences about
gene therapy safety. To probe the specificity of the ZFP for the target DNA-
binding site, in vitro binding profiles are experimentally derived [62]. Then,
in silico prediction can be used to determine a number of predicted off-target
sites to be PCR amplified. The Surveyor assay may be used to evaluate off-target
cleavage at the predicted loci. Because this technique is limited by the successful
prediction of the off-target binding sites, large-scale sequencing methods may
be preferable as they provide a more comprehensive view of all off-target events
in the genomic DNA [63, 64]. Whole-exome sequencing and next-generation
sequencing methods have become widely available and more commonplace in
recent years [65]. These sequencing methods permit unbiased whole-genome
analysis of ZFN specificity. However, small numbers of off-target events may
not be effectively found by this or any method, so practical assessment of the
transformation and clonal expansion of treated cells may be performed by
established methods such as a soft agar assay are also advisable for development
of ZFN-based clinical products [66].
3.5 Methods for Delivering Engineered Zinc Finger
Nucleases into Cells
The ability to perform targeted genome modification using ZFNs is dependent
on the delivery of the ZFNs into target cells and into the nucleus. The ZFN genes
may be introduced into the cell by viral or nonviral methods. Nonviral meth-
ods to transfect cells ex vivo include lipophilic reagents and electroporation.
Electroporation of plasmid DNA or RNA has proven effective, though elec-
troporation can be toxic to cells and is less efficient than other methods such as
viral delivery [67]. Viral delivery has been successful, including adenovirus [59],
integrase-defective lentivirus [68], and adeno-associated virus [69–71]. Viral
delivery can be used for both the delivery of the ZFN and homologous DNA if
HR-directed modifications are desired. More recently, ZFN protein has been
shown to be capable of traversing cell membranes to achieve genome editing
[72]. Ultimately, the delivery methodology use for ZFN-mediated genome modi-
fication will depend on the cell type targeted and whether or not the cells will be
modified in vitro or in vivo.
3.6 Zinc Finger Fusions to Transposases and Recombinases
ZFNs comprise the more characterized class of proteins for site-directed genome
modification. However, all nucleases, including ZFNs, have serious limitations.
Difficulties in measuring the rates of off-target DNA cleavage, the dependence
on cellular DNA repair machinery, high levels of DSB-induced toxicity leading to