3.1 ntroduction to Zinc Finger DNA--inding Domains and Cellular epair Mechanisms 35proven to be very useful for the genomic manipulation of yeast, where rates of
HR are naturally high [4]. In contrast, mammalian cells provided with a homolo-
gous DNA template have extremely low background rates of HR: only one in a
million somatic cells shows evidence of HR-mediated repair following homolo-
gous DNA introduction [5, 6]. To stimulate repair, DSBs may be introduced to
encourage the cell to repair the DNA using the abundantly available homologous
DNA template [7]. ZFN-induced DSBs stimulate HR at target sequences. In this
way ZFNs may be used to introduce or correct point mutations in a seamless
manner without additional sequences, making this strategy preferred to gene
addition strategies when possible. Unfortunately, the frequency of HR is cell-type
dependent and cell division is required, ruling out many potential applications
[8, 9]. Despite the limitations, this method has been applied for genomic manip-
ulation of many species across the kingdoms of life, including bacteria, yeast,
plants, and mice, as well as in human cells for seamless gene correction [10].
3.1.3 Non-homologous End Joining
NHEJ is a system of DSB repair that acts by directly ligating the ends of linear
DNA. If the break is staggered and homologous sequences exist on either side, an
accurate repair can be made by sensing the homology [11]. However, if there are
no homologous strands, NHEJ can still mediate repair to stitch back together the
DNA via the protein Ku70 [10, 12]. In this case, there is usually the gain or loss of
a few base pairs of DNA resulting from the chemical repair of the free ends of the
DNA. Under highly stressful conditions such as ultraviolet radiation, toxins,
radioactivity, or desiccation, the cell could suffer multiple DSBs. In this case,
when there are more than two free linear ends, the cell may ligate the incorrect
ends together, resulting in chromosomal rearrangements or large deletions. Such
major insults can result in a cancer-causing phenotype or more likely cell death.
Even with this possibility, NHEJ is still highly conserved due to the huge advan-
tage to the organism of having a DNA repair mechanism that does not rely on the
presence of homologous sequences on the sister chromatid.
Gene deletion or addition can be achieved with NHEJ, while the elegant seam-
less gene correction or addition strategies require HR. The majority of the cell
types comprising an adult human, which are the cells that are most desirable to
be targeted for correction in a gene therapy setting, prefer NHEJ over HR. In
transfected cells, using I-SceI to induce a DSB and the sister chromatid for HR
rarely results in gene correction, making NHEJ the easier goal to achieve.
Demonstrating this point, a clinical trial has been completed using a ZFN to
knock out the CCR5 receptor to block HIV infection [13, 14], while there are no
clinical trials underway that rely upon HR-mediated gene correction.
Either of these repair strategies can be exploited using ZFNs for targeted
genomic modification. NHEJ can be used for targeted mutagenesis of chromo-
somal elements [15]. HR can be used for targeted DNA repair or gene addition
by providing a template strand of DNA homologous to the targeted site of DNA
cleavage [16–18]. Therefore, targeted DNA cleavage can be used to achieve tar-
geted mutagenesis, targeted DNA repair, or targeted DNA addition at specific
genomic sites.