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Zinc fingers hit off target
© 2011 Nature America, Inc. All rights reserved.
There has been a lot of excitement about the potential
applications of zinc finger nucleases (ZFNs), as these
enzymes allow highly specific, targeted genome modification
in live cells. ZFN-mediated gene correction is more specific
than the viral integration methods currently used in gene
therapy, and two ZFNs are currently in clinical trials for
treating HIV and cancer. However, two new papers by
Pattanayak et al.1 and Gabriel et al.2 raise questions about
the purported specificity of these genome modification tools.
They show that ZFNs, in addition to cleaving at their desired
sites, can also have unexpected cleavage effects in vivo that
cannot be predicted using conventional in silico analyses.
These findings could have important consequences for the
safe use and optimization of ZFNs in gene therapy. We asked
four experts to comment on the implications of this study.
Linzhao Cheng
These two studies1,2 are important and timely, providing a
sobering assessment of the reality of ZFN-mediated gene targeting. Although it is disappointing (but not surprising) to realize the inherent imperfections of this method, there are several
ways we could avoid off-target effects and optimize targeting
in human cells. For example, screening in vitro1 or in a model
cell line2 is better than in silico predictions of all the possible
ZFN cleavage sites on the basis of biophysical and biochemical principles, although not all the potential sites will be cut in
a given cell type, especially if the ZFN concentration is optimized. Therefore, the most relevant cell types should be used
for gene targeting, and the targeted cells should be analyzed
before conducting important biological experiments or putting
the targeted cells into patients.
It is unlikely that we will be able to completely avoid offtarget events in the near future, even if the specificity of ZFNs
or related technologies such as homing endonucleases or transcription activator–like effector nucleases (TALENs) is further
improved. However, for most applications, we will be able to
select correctly targeted cells without needing to ensure correct targeting in every cell. In fact, current gene targeting technologies using homologous recombination rely on the ability
to select and expand rare cells that have been correctly gene
targeted, because the homologous recombination rate in nontransformed human cells is still low (1 × 10-4 or less), even when
aided by ZFNs. Therefore, it would be desirable to use cell types
that can be substantially expanded in culture, such as stem cells.
Human pluripotent stem cells make it feasible to select a correctly targeted clone3,4. With the improved efficiency of gene
targeting achieved by ZFNs and other tools, and the improved
capacity for whole-genome DNA sequencing of several selected
clones, we should be confident that it will be possible to derive
precisely edited human cells for basic research and gene therapy.
Professor of Medicine, Stem Cell Program in the Institute
for Cell Engineering, The Johns Hopkins University School
of Medicine, Baltimore, Maryland, USA.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
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Bruce Blazar
ZFNs are reagents that allow precise gene targeting and offer an advantage
over gene therapy vectors that are prone to semirandom genomic
integration. Given that the majority of the genome is transcribed5 and thus
may produce either protein-coding or regulatory transcripts, it is of the
utmost importance that only the intended genomic sequence is targeted.
“Even these precise tools can cause
unintended genomic modification.”
Two recent manuscripts analyzed the off-target effects of ZFNs and
show that even these precise tools can cause unintended genomic
modification1,2. The papers overlap in their analysis of a ZFN targeting the
chemokine receptor gene CCR5 that has already entered clinical trials as
an anti-HIV therapy. Using an in vitro DNA-binding assay, Pattanayak et
al.1 identified 37 sites in the human genome cleaved by the CCR5 ZFN.
Analysis in intact human cells, however, showed that only ten of the sites
were actually modified in K562 cells. Using the same cell type and ZFN
but a different approach, whereby an integrase-deficient lentiviral vector
cassette can be ‘trapped’ in a ZFN-induced double-stranded DNA break,
Gabriel et al.2 mapped the genomic sites at which the ZFN was active.
They showed that 93.8% of the insertion events were at the CCR5 locus.
Interestingly, in only one case did both studies identify the same off-target
site, CCR2. Given that in vitro DNA binding does not fully predict in vivo
binding and given the high homology between the two genes, the finding
that the CCR2 locus was affected is not surprising.
Collectively, these studies offer guidance for future ZFN design and
use and suggest that in silico analyses may be best suited toward reagent
Katherine High
Pattanayak et al.1 and Gabriel et al.2 deficient lentiviral vector to tag CCR5
have reported two different approaches ZFN target sites in K562 cells and then
to identifying off-target cleavage sites of mapped the sites using linear amplificaZFNs. These papers invite comparison, tion-mediated polymerase chain reacas they both examtion (LAM-PCR)
ine, in the same cell
and sequencing
“An issue that
line, a pair of ZFNs
approaches. By conremains to be
that cleave at the
trast, Pattanayak
CCR5 locus, and
et al.1 screened a
addressed is when
one might predict
library of 1 × 1011
during clinical
that they would
DNA sequences
identify similar or
to identify those
development offoverlapping groups
that were cleaved
target sites need to be
of off-target sites.
by the CCR5 ZFNs
Unexpectedly, the
in vitro. They then
thoroughly analyzed.”
papers do not seem
examined 34 potento agree, but because they use different tial cleavage sites found in the human
analyses and because off-target sites are genome by deep sequencing them from
sufficiently rare, this is perhaps not so transfected K562 cells expressing the
surprising.
ZFNs. Differences in the sequences of
Gabriel et al.2 used an integrase- the CCR5 ZFNs, and in their concenvolume 17 | number 10 | october 2011 nature medicine
com mu n i t y co r n e r
© 2011 Nature America, Inc. All rights reserved.
Kenneth Eward / Photo Researchers, Inc.
Matthew Porteus
optimization rather than prediction of off-target effects. Because ZFNs
can bind with some degree of mismatching, off-target effects can occur,
which may be minimized by using lower ZFN concentrations and optimizing
binding to the on-target site1,2. As this field of research progresses, factors
that determine ZFN target specificity will become better defined. Finally,
direct testing of ZFN reagents that have optimized architectures in a
nonbiased manner in the cell type of interest will be crucial for future ZFN
clinical applications.
Regents Professor and Andersen Chair in Transplantation Immunology,
Department of Pediatrics, Division of Blood and Marrow Transplantation
University of Minnesota, Minneapolis, Minnesota, USA.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
trations in the cell lines, may partially
account for the differences in identified
off-target sites1,2.
For clinical applicability, an unbiased analysis of cleaved sites following
exposure to ZFNs in a way that most
closely mimics the desired application
is important. It is unclear what would
be gained by in vitro analysis of a DNA
library, when off-target cleavages that
occur in endogenous DNA that is subject to dynamic epigenetic modification
are more relevant to clinical studies. Therefore, the analysis by Gabriel
et al.2, and a similar one recently
reported6, seem to model potential
clinical application more faithfully.
The latter report, in which ZFNs were
expressed in mice6, allows collection of
in vivo safety data over many months,
which can supplement the molecular
analyses described in these two reports.
An issue that remains to be addressed
is when during clinical development
off-target sites need to be thoroughly
analyzed. Off-target sites that threaten
cell viability will be identified in early
experiments, but the clinical effects of
rare events with subtle effects may be
more difficult to identify.
Investigator, Howard Hughes
Medical Institute, Children’s Hospital
of Philadelphia, Philadelphia,
Pennsylvania, USA.
COMPETING FINANCIAL INTERESTS
The author declares competing financial
interests: details accompany the full-text
HTML version of the paper at http://www.
nature.com/naturemedicine/.
Since the first report of using ZFNs for targeted genome
modification in mammalian cells, they have been known
to cause cellular cytotoxicity7. First-generation assays for
cellular toxicity used cell viability and immunofluorescence
of DNA double-strand breaks to measure the specificity of
ZFNs. These effects arose from ZFNs cutting at specific offtarget sites in the genome, but the identity of these sites was
not known.
Importantly, Pattanayak et al.1 and Gabriel et al.2 have
now developed different approaches to identify some of
these sites. Interestingly, using the same ZFNs in the same
cell line and excluding the obvious CCR2 site, the two
groups identified a non-overlapping set of sites. Thus, the
identification of the complete set of off-target sites, even for
the well-studied pair of ZFNs used in these studies, is not
complete. Both studies highlight that previous approaches
using in silico prediction methods are of limited utility, and
claims of ZFN specificity on the basis of such in silico studies should be reevaluated. Moreover, the strategy each group
used to validate their off-target sites was focused on the
creation of small insertions and deletions rather than the
rarer, but potentially more problematic, gross chromosomal
rearrangements. Thus, future studies will need to focus on
both the identification of a more comprehensive set of offtarget sites and the frequency with which ZFNs create gross
chromosomal rearrangements.
Ultimately, the functional significance of off-target
effects of ZFNs rather than the frequency of these events
will need to be assessed, as the simple creation of doublestrand breaks, which are caused by many widely used therapies, does not preclude the clinical use of these enzymes.
Finally, Pattanayak et al.1 discuss that precise expression of
ZFNs (the ‘Goldilocks’ effect) is essential to maximize ontarget activity while minimizing off-target activity. Small
molecules have already been used to regulate ZFN protein
expression with this concept in mind, and this is just one
approach for titrating ZFN expression so it’s ‘just right’8.
Associate Professor, Department of Pediatrics, Divisions of
Cancer Biology, Hematology/Oncology and Human Gene
Therapy, Stanford University, Stanford, California, USA.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
1. Pattanayak, V., Ramirez, C.L., Joung, J.K. & Liu, D.R. Revealing off-target cleavage specificities of zinc-finger nucleases by in
vitro selection. Nat. Meth. 8, 765–770 (2011).
2. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).
3. Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.
Nat. Biotechnol. 27, 851–857 (2009).
4. Zou, J. et al. Oxidase deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by
zinc finger nuclease mediated safe harbor targeting. Blood 117, 5561–5572 (2011).
5. ENCODE Project Consortium et al. Identification and analysis of functional elements in 1% of the human genome by the
ENCODE pilot project. Nature 447, 799–816 (2007).
6. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011).
7. Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).
8. Pruett-Miller, S.M., Reading, D.W., Porter, S.N. & Porteus, M.H. Attenuation of zinc finger nuclease toxicity by small-molecule
regulation of protein levels. PLoS Genet. 5, e1000376 (2009).
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