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special feature | COMMENTARY
method of the year
Zinc-finger nucleases: how to play two good hands
Mark Isalan
© 2012 Nature America, Inc. All rights reserved.
Zinc-finger nuclease dimers are more difficult to engineer than single DNA-binding domains, but the
development of new methods could help.
The potential of gene targeting with
zinc-finger nucleases (ZFNs) was first
explored by the groups of Carroll and
Chandrasegaran1 and has resulted in the
explosive growth of an exciting new field.
In the typical implementation, pairs of artificial zinc-finger DNA-binding proteins
(ZFPs) containing three or four zinc fingers apiece are linked to the FokI nuclease
domain to create a sequence-specific nuclease upon dimerization (Fig. 1). The nuclease can then be used to mutate chromosomal targets, via a double-stranded DNA
break, either by error-prone nonhomologous end joining (NHEJ)2 or by stimulation
of homologous recombination with a donor
DNA template3.
Despite the growing number of publications attesting to the power of this method,
there is an elephant in the room: it is very
difficult for the average research group to
build functional ZFNs for particular target
DNAs. Successful projects either buy readymade ZFNs, employing the enormous
resources of companies such as SigmaAldrich (CompoZr ZFNs), or collaborate
with academic teams, using the opensource tools of the Zinc Finger Consortium
(http://www.zincfingers.org)4. Commercial
ZFNs were once expensive custom projects
but now also include cheaper off-the-shelf
reagents (although to use these one may
need to compromise on the specific target
site). Alternatively, the consortium provides
convenient computational tools such as
ZiFiT5 to help design constructs, although
this can still be challenging for inexperienced users; building one or two ZFNs à la
carte can often result in failure.
EMBL/CRG Systems Biology Research Unit, Centre
for Genomic Regulation and UPF Barcelona, Spain.
e-mail: [email protected]
To G or not to G
The reasons that ZFNs are challenging to build are outlined in Figure 1.
The major limiting factor is that it is not
possible to target just any desired DNA
sequence. Although zinc-finger engineering has been extensively developed 6,
it is no coincidence that the zinc fingers used as engineering scaffolds have
G-rich consensus sequences (for example,
Zif268: 5ʹ-GCG(G/T)GGGCG-3ʹ; Sp1:
5ʹ-GGGGCGGGG-3ʹ). My group and
others have put great effort into targeting
non–G-rich sequences (for instance, by
using overlapping finger approaches 7,8),
but the fact is that G-rich sequences are
the natural preference of zinc fingers.
Assembly of fingers for other types of targets is frequently unsuccessful 9, for two
reasons. First, the strongest protein-DNA
interactions are between arginine residues
and guanine bases in the major groove10.
Second, overlapping contacts between
adjacent fingers have a configuration in
which guanines are particularly stable at
the first and third bases of each fingerrecognition site11. Therefore, perhaps the
best advice for novice ZFN engineers is to
choose a G-rich target from their computer-generated list of hits.
This G preference puts a real constraint
on the specific sequences that can be
targeted by ZFNs, and it is related to the
second problem: it is far easier to make
a single designer DNA-binding domain
than to join together two domains, in the
appropriate orientation, with the correct spacing, to yield a functional nuclease. Given 1 kb of genome sequence, it is
relatively straightforward to find a suitable patch to target the binding of a single ZFP domain, and this has been done
routinely for over a decade 6 . However,
32 | VOL.9 NO.1 | JANUARY 2012 | nature methods
for nucleases, the ‘easiest’ sequence to
target is 5ʹ-MNNCNNCNNCNNCX 5–
7 GNNGNNGNNGNNK-3ʹ (where M
= A,C; N = contacted bases (ideally G
rich); X = uncontacted bases; and K =
G,T), a consensus sequence derived from
structural considerations and a wealth of
phage-display experiments6. Furthermore,
the ideal target will be unique in the
genome, with few closely related sequences that could potentially draw off-target
binding (targets and ‘off-targets’ should be
at least 3 bp different and share less than
two-thirds homology12,13).
As can be seen, therefore, even before
one begins to engineer a ZFN, target selection is quite restricted. It is best achieved
with a web-based computer program5.
Gaps, linkers and cutting-site position
The 5- to 7-bp gap in the target between
the sequences recognized by the two ZFP
domains is another constraint for ZFN
engineering and is determined by the
design of the linker between each ZFP
and the FokI nuclease. The constraint
can be relaxed by using much longer
linkers, but this comes at the expense of
increasing binding promiscuity resulting
from accommodating many different gap
lengths1. For most applications, restrictive
linkers are preferred: -LRGS-4 (5- to 6-bp
gap), -QNKK-1 (6-bp gap) and -TGQKD(7-bp gap; K.A. Wilson and M.H. Porteus,
personal communication) are all linkers
that fit between the ZFP terminal histidine
and the -QLV beginning of FokI.
The restrictions on target choice have a
bearing on the final application of the ZFN
because it is sometimes desirable to target
an exact base position. For example, for the
purpose of integrating foreign DNA efficiently into a locus, the inserted cassette
COMMENTARY | special feature
ZFPs functional as a pair
G-rich
target
Correct finger
overlap
Appropriate linkers
5–7-bp gap
-N
FokI
C-
M NN CNNC NN CNNC
K NN GNNG NN GNNG
Specificity,
affinity,
kinetics
NN NNNN
NN NNNN
FokI
G N N GN N GN N G N N K
CNNCNN C NNCNN M
-C
Improved FokI mutants
N-
Precision of cleavage site
Number of fingers
Unique DNA target in genome, no related sites, accessible chromatin
© 2012 Nature America, Inc. All rights reserved.
Figure 1 | Overcoming the challenges for engineering functional zinc-finger nucleases. The schematic
shows two four-finger pairs binding to DNA in a canonical mode and highlights the main engineering
constraints and considerations. Zinc-finger binding sites are shaded in blue (darker for the main
contacted DNA bases).
should be introduced at the exact site of
the ZFN cut, in between the two homology arms14. Exact positioning is required
here because it is thought that the cleaved
chromosomal DNA ends need to prime
on the donor template DNA, so a nonadjacent integration cassette would not be as
effective.
For introducing or correcting point
mutations by homologous recombination,
there is slightly more flexibility. The mutations should still be as close to the cutting
site as possible but can be anywhere within about 400 bp, as most recombination
events will occur within this distance 15.
It is also worth noting that one can introduce a few silent mutations on the donor
plasmid (mutating guanines) to prevent
ZFN cutting of the template DNA. Finally,
introducing a knockout mutation by errorprone NHEJ (using ZFNs in the absence
of a donor DNA) provides even more flexibility in cutting-site position. Cutting
sites, and hence frame-shifting mutations,
can be put anywhere in a coding sequence,
although more N-terminal positions are
usually preferable.
Targeted libraries
A consistent source of confusion in zincfinger design is the fact that zinc fingers
defy convention and bind DNA ‘backward’, with the zinc finger N–C binding
DNA 3ʹ–5ʹ. Therefore, one must constantly specify DNA direction, and it
helps to ‘think’ in the 3ʹ–5ʹ direction when
designing fingers and libraries. Library
design and engineering strategies are too
numerous to describe here but have been
discussed at length elsewhere4,6. Briefly,
the main restriction in building libraries
to make new DNA-binding zinc fingers is
a combinatorial problem: the number of
possible variations in the DNA recognition helices rapidly increases beyond what
is practical to screen, even when only five
or six amino acid positions are randomized. Existing methods overcome this in
various ways, for instance by engineering smaller parts bit-by-bit, resulting in
validated archives of one- or two-finger
modules. Although the modern user can
simply use a computer-generated archive5
to build ZFNs from parts, they are still left
with a problem when a ZFN simply fails
to work.
When fingers fail, in vitro cutting
assays16 can rapidly identify whether one
or both halves of the nuclease are working, and this can guide a researcher as to
what to do next. When both halves fail
it is probably simplest to choose another
target site. However, when only one half
of the nuclease fails, it is very tempting to
build a mini-library to screen for variants
that rescue the failed half-ZFN. Although
there are several ways to do this, including
dedicated bacterial one-hybrid17 and twohybrid systems4, my group reasoned that
it would be convenient to have an option
that uses commercially available components. We therefore recently adapted a
commercial yeast one-hybrid kit to select
zinc fingers from targeted mini-libraries
and used this to rescue ‘orphan’ half-ZFNs,
resulting in working ZFN pairs16. The key
decisions for success in this type of effort
relate to how one achieves limited randomization of the finger framework.
The size of the library in such a screen
for active ZFP monomers is dependent on
the number of fingers in the construct: the
more fingers there are, the more difficult
it is to randomize, as larger libraries are
required. Longer chains of zinc fingers
will also have an impact on the affinity and
interaction kinetics of the DNA-binding
domains. Longer ZFPs have increased
affinity but slow off rates, with half-lives
of days (see references in ref. 6), and simply adding more fingers does not appear
to make the best ZFNs. For instance, an
analysis of off-target cutting has suggested
that avoiding ZFPs with very high binding energy could improve overall specificity12. Intriguingly, it was recently reported
that three- or four-finger pairs can cut
better than five- or six-finger pairs 18 .
Nonetheless, there are several examples in
the literature in which five- or six-finger
ZFN pairs have been used successfully,
albeit with longer (six-amino-acid) linkers after every second finger (for example,
TGSERP rather than canonical TGEKP).
Noncanonical linkers can even be used
to jump bases between finger subsites,
but because this option is not included in
programs such as ZiFiT, it is best avoided
by the average user. As with other aspects
of ZFN engineering, success depends on
being careful with the details.
Improving efficiency
Protein design and library selection have
not been limited to the DNA-binding
domains of ZFNs. For several years now,
obligate heterodimer mutations 19,20 in
the FokI domain have been the constructs of choice, preventing promiscuous cutting from unwanted homodimers.
More recently, a FokI ‘Sharkey’ mutant
was engineered for improved activity 21.
Other refinements to increase efficiency
include using 30 °C cold shock to enhance
NHEJ 22, using alternative promoters to
express ZFP (for example, the phosphoglycerate kinase promoter, pPGK16), using
replicating plasmids (with SV40 origins of
replication and large T antigen), and waiting for 7 days after ZFN and donor plasmid transfection before assaying homologous recombination.
A recent innovation uses an elegant
trick to enrich for ZFN-modified cells
indirectly23 (Fig. 2a). Co-transfection of
an episomal reporter plasmid with a ZFN
site between the coding sequences of an
RFP and an out-of-frame GFP means
that cells in which there was more effective ZFN activity show green fluorescence
because the GFP is restored via NHEJ.
These cells can be recovered by FACS. As
the system uses transient transfection,
the reporter dies away rapidly, leaving
marker-free cells. Thus, the process can be
nature methods | VOL.9 NO.1 | JANUARY 2012 | 33
special feature | COMMENTARY
© 2012 Nature America, Inc. All rights reserved.
Figure 2 | Screening for full zinc-finger
nuclease activity. (a) In a scheme
developed by the Kim group23, the
fluorescence of an out-of-frame GFP
is restored by a functional zinc-finger
nuclease (ZFN). This was originally
designed as an indirect marker for FACS
of ZFN-modified cells. However, it could
be adapted to screen candidates from
ZFN libraries in mammalian cells, as
schematized here. (b) For screening
larger ZFN libraries in a eukaryotic
chromatin environment, the yeast onehybrid system we recently described16
could be adapted. ZFN cleavage of a
negative selection marker (URA3 +
5-FOA) would be required for growth
on a plate. NHEJ, nonhomologous end
joining. Fokl+ and Fokl– are obligate
heterodimer variants19.
a
repeated for several rounds23 to enrich for
genome-modified cells progressively, even
using ZFNs with relatively low efficiency.
The existence of such a generic eukaryotic screen for ZFN activity raises the possibility of screening randomized libraries
of ZFNs directly in mammalian cells. As
long as library sizes can be maintained
within practical limits, an advantage
of testing the ZFPs in their paired context would be the elimination of orphan
half-ZFPs. Another advantage is that one
would be testing in a more realistic chromatin environment, as the in vitro and in
vivo activities of these nucleases do not
always correspond 4. However, mammalian systems based on transient transfection would not allow the partitioning of
one library member per cell, and so transfections would have to be carried out oneby-one in well formats (Fig. 2a).
One c ou ld a lter nat ively imag ine
adapting our yeast one-hybrid system 16
to screen for nucleases (Fig. 2b). This
would have several potential advantages:
full nuclease pairs would be selected in a
eukaryotic chromatin environment (albeit
in yeast), low-copy yeast plasmids would
segregate as one library member per
cell, and well-established yeast selection
markers would allow for positive selection of transformants and counterselection of cells with inactive ZFN variants.
One could begin with a lead ZFN design
from ZiFiT and mutate around the DNAbinding helices 16 to screen directly for
ZFN cleavage. The selective principle outlined in Figure 2b exploits the selective
toxicity of 5-fluoroorotic acid (5-FOA)
when the URA3 uracil biosynthesis gene
ZFN library member
b
PCR ZF library–L
Stop
*
RFP
Target
site
PCR ZF library–R
FokI+
Out-of-frame
GFP
FokI–
TRP1
NHEJ
Prey
plasmids
LEU2
ZFN excises URA3
STOP
In-frame
GFP
Target site
URA3
HIS3
Target site
Bait
plasmid
Co-transform yeast and plate on
SD _His _Leu _Trp + 5-FOA
FACS sort and
identify ZFN
Positive selection for triple transformants (HIS3, LEU2, TRP1);
negative selection for URA3 not excised by ZFN
is active: only active ZFNs would excise
URA3 and allow cell survival in medium
containing 5-FOA. I emphasize that this
is a thought experiment—which we currently have no intentions of implementing—but it would be interesting to see
whether full ZFN-pair selection would
lower failure rates in vivo.
A tell-TALE sign of things to come
As a multitude of methods exist for engineering zinc fingers, one could be forgiven
for thinking that making ZFNs is a solved
problem. I have tried here to redress the
balance by highlighting the fact that there
are still many issues that need to be considered on a case-by-case basis. But it should
not be forgotten that when ZFNs do work,
they work very well3, and so they should
not be sidelined too quickly, even if there
are promising new technologies such as
meganucleases24 and transcription activator–like effector (TALE) nucleases on
the horizon25. The latter are being rapidly
embraced by the research community, and
ZiFiT was recently updated (version 4.0) to
allow TALE nuclease design5. Will TALE
nucleases be the solution to the G problem
and become the nucleases of choice? What
will their specificity and toxicity profiles be
like in vivo? Alternatively, will commercial
sources of ZFNs become so accessible that
no custom academic projects will need to
be undertaken? Only time will tell.
ACKNOWLEDGMENTS
M.I. is funded by the European Research Council,
FP7-ERC-201249-ZINC-HUBS, Ministerio de Ciencia
e Innovacion grant MICINN BFU2010-17953, and
The Ministerio de Educacion y Ciencia and European
Molecular Biology Laboratory (MEC-EMBL) agreement.
34 | VOL.9 NO.1 | JANUARY 2012 | nature methods
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
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