42 3 Site-Directed Genome Modification with Engineered Zinc Finger Proteins
cell death, and the requirement for cell division are just some of the problems
caused by inducing free DSBs in the cell. DSBs are associated with carcinogenic
agents and can cause undesired chromosomal translocations [73], although there
have not been any reports to date of ZFNs causing cancer. Enzymes such as
recombinases and transposases are capable of DNA excision and integration
autonomously, without free DSBs and their negative aspects. However, trans-
posases require very short sequences for integration, usually 2–8 bp, making
their integration essentially random [74]. Fusion of ZF DNA-binding domains to
recombinases [75–77] and transposases [78, 79] has resulted in successful redi-
rection of the integration events to varying degrees. Recombinases have built-in
DNA specificity and thus require reengineering to target user-defined chromo-
somal targets [75–77]. Transposase fusions do not require such engineering
since their target sites are so short. ZF–transposase fusions are sometimes highly
active [78, 80, 81]. However, these systems require further refinement. Firstly,
transposase fusions have not yet demonstrated a high level of specificity in
genomic targeting because the transposase portion of the ZFP transposes in a
manner that is independent of the ZF DNA-binding domain. In order to increase
specificity, one idea is to mutate the ZF–transposase fusions such that the trans-
posase domain is kept inactive until the ZF portion binds the DNA. Secondly,
transposase ZFPs require the presence of their short transposase target site in
close proximity to the site recognized by the ZF [79], placing a limit on the avail-
able target sites. Finally, despite many advances, effective engineering of a ZFP to
target a unique genomic locus has not yet been accomplished. Attempts to target
the checkpoint kinase-2 (CHK2), the ROSA26 locus, and the l-gulono-γ-lactone
oxidase pseudogene (GULOP) were unable to produce successful targeting in
cells [79, 82]. Further development of proteins other than ZFNs for genomic
targeting should lead to diverse technologies capable of site-specific gene addi-
tion, even in cells not actively dividing.3.7 Conclusions
ZFNs are a proven tool for targeting endogenous loci in the genome, while ZFPs
have the potential for user-defined modification of chromosomal targets without
DSBs. Over the years “open” access to ZFP engineering tools together with com-
mercial availability led to more widespread use. However, while ZFNs and other
nucleases began the field of targeted genome modification, one might expect for
the focus on ZFNs to decrease in the coming years as the ease and simplicity of
working with the CRISPR/Cas9 system displaces the older, more expensive, and
time-consuming ZFN platform. The Cas9 system can attribute the exponential
pace of its development to the established systems and assays that were devel-
oped for engineering and testing ZFNs. As clinical trials usually take over a dec-
ade to reach the clinic and the ZFNs have a different set of patents governing
their use, it is still quite possible that ZFN-based drugs could become approved
for therapeutic use at some point in the near future. ZFPs will continue to be
important tools for genome engineering to ask critical biological questions as
well as development of novel therapeutics to improve human health. Time, and