38 3 Site-Directed Genome Modification with Engineered Zinc Finger Proteins
tools for evaluating for ZFN target sites within a genomic DNA region for both
OPEN and CoDA at the http://www.zincfingers.org website. Because OPEN and CoDA
rely upon previously validated ZFPs, there are some sequences that cannot be
targeted using these methods. Based on the failure rates and time investment for
each approach, investigators should first consider CoDA, then OPEN, and then
modular assembly only if the target sequence is unavailable through CoDA or
OPEN. Supporting this recommendation, recent computational studies have
suggested that binding of the ZFP to the DNA sequence is better thought of as
synergistic rather than strictly modular [49, 50].3.2.3 Purchase via Commercial Avenues
Engineered ZFPs are also available commercially. Sangamo Therapeutics, Inc.
developed a proprietary archive of engineered ZFs early on but has not made
this information public, although they have published some of the details regard-
ing their ZFN engineering platform [48]. Currently, the simplest means of
obtaining a ZFN pair to a novel target sequence is by purchasing a custom pro-
tein. Sangamo licensed its proprietary methodology to Sigma-Aldrich, which
has marketed the technology as the CompoZr Zinc Finger Nuclease platform.
Pre-validated ZFNs to the rat and mouse Rosa26 locus as well as the human
AAVS1 safe harbor site present the most cost-effective option. These would
allow the investigator to place transgenes at known genomic locations that will
not interfere with genomic function, are commonly used, and are known “safe
harbor” sites. Additionally, ZFNs to target an abundance of specific human,
mouse, and rat genes are available at a more reasonable cost as compared with
custom target options; the complete list of the genes is available online at http://www.
sigmaaldrich.com. Custom ZFNs designed to target novel sequences require
increased time and are produced at a much greater cost. A major advantage of
using a commercial service to design a custom ZFN is the timeframe of delivery
in less than 3 months. For most research investigators, use of the clustered regu-
larly interspaced short palindromic repeat (CRISPR)/Cas9 system of targeted
integration is now the fastest and most cost-effective method by which to initi-
ate a nuclease-driven project [10]. Later on, purchased or designed ZFNs may be
integrated into the molecular toolkit for intellectual property, reproducibility, or
other experimental reasons.3.3 Genome Modification with Zinc Finger Nucleases
Engineered ZFNs can be used for a variety of genome alterations. These can be
categorized as dependent on either HR or NHEJ. HR-based alterations include
targeted addition of DNA sequence to the genome [16–18] through introduction
of a new sequence flanked by homologous arms or targeted base-pair changes
achieved by supplying a homologous template with the desired alteration. NHEJ-
based changes do not require the introduction of homologous sequences and
include gene disruption strategies that take advantage of the infidelity of NHEJ
repair mechanisms. Another NHEJ strategy involves supplying ZFN pairs for