Advances in Zinc Finger Nuclease and Its Applications

Review
Gene and Gene Editing
Copyright 2015 American Scientific Publishers

All rights reserved
Printed in the United States of America
Vol. 1, 315, 2015

www.aspbs.com/gge
Advances in Zinc Finger Nuclease and Its Applications

Li-Meng Tang1 , Cui-Lan Zhou1, Zi-Fen Guo1 , Li Xiao2 , and Anderly C. Cheh3
1
Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China
Laboratory of Molecular Medicine, The Second Affiliated Hospital of Soochow University, Suzhou 215123, China
3
Department of Medicine (Austin Health), The University of Melbourne, Parkville, VIC 3052, Australia
2
Zinc finger nuclease (ZFN) is an artificially engineered hybrid protein consists of a series of zinc finger protein domains,
fused to a cleavage domain of Fok I endonuclease. As a powerful molecular tool for gene editing, ZFN has been widely
used for gene knock-in and knock-out in a variety of cell types and organisms. However, due to the limitations of its
specificity, technological improvement for ZFN and its applications are much needed. This review summarizes the latest
improvements of such technique for use in biology.
KEYWORDS: Zinc Finger Nuclease, Gene Editing, Off-Target Effect.
CONTENTS
eukaryotic zinc finger-containing proteins (ZFPs) that
Structure of ZFNS and Their Mechanism of Actions in

exhibitdeanSantiago
unique ability
of recognizing and binding to
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de Chile
Gene Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
specific
DNA
sequences.
The
series of zinc finger protein
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Applications and the Current Limitation of ZFNS . . . . . . . . .
6
Copyright: American
Scientific
Publishers
domains
is
also
termed
zinc
finger
motifs or zinc finger
Disease Modeling at Cellular Level or
arrays. Based on the conserved amino acids in the zinc
Intact Organismal Level . . . . . . . . . . . . . . . . . . . . . .
6
finger domain, ZFPs and ZFNs can be categorized into
Genome Modification of Organisms for
Agriculture and Industry . . . . . . . . . . . . . . . . . . . . . .
6
three major subtypes (C2 H2 , C4 , and C6 ).1 Among these,
Human Gene Therapy . . . . . . . . . . . . . . . . . . . . . . .
7
C2 H2 is the classic type and the most widely used in the
Current Limitations of ZFNs . . . . . . . . . . . . . . . . . . .
8
artificially-engineered
ZFNs. TFIIIA was the first identiAdvances in ZFN Cloning . . . . . . . . . . . . . . . . . . . . . . . .
8
fied C2 H2 ZFP that consists of with zinc finger domain of
Optimization of the Zinc Finger Binding Domain . . . . . .
8
30 amino acids, in which the 8th and 13th amino acids
Optimization of the Reporting and Screening System . . . 10
Modification of the Cleaving Domain of Fok I . . . . . . . . 10
of cysteine and the 26th and 30th amino acids of hisOptimization of ZFN Modification Technology . . . . . . . 11
tidine are highly conserved throughout evolution. These
Optimization of Cell Transfection Technology . . . . . . . . 11
conserved amino acids are responsible for the direct bindOff-Target Effects of ZFN . . . . . . . . . . . . . . . . . . . . . 11
ing to zinc ion,2 which critical in stabilizing the protein
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
fold for DNA binding. This type of Cys2His2 (C2 H2 ) ZFP
STRUCTURE OF ZFNS AND THEIR

MECHANISM OF ACTIONS IN GENE EDITING
Zinc finger nuclease (ZFN) is a class of artificiallyengineered nuclease that consists of a series of zinc finger
protein domains fused with the cleavage domain of nuclease Fok I (Fig. 1). The sequence specificity and binding
affinity of ZFNs to the target DNA are determined by zinc
finger protein domains derived from naturally-occurring
Author to whom correspondence should be addressed.

E-mail: lixiao0626@sina.com
Received: 10 January 2014
Accepted: 6 May 2014
Gene Gene Edit. 2015, Vol. 1, No. 1
typically binds to DNA in the form of monomer. By comparion, the type of C4 ZFP has 4 cysteines in as the conserved amino acid, which binds to DNA in a form of
dimer. Reversed repeats in target DNA sequence are recognized by homodimer, whereas direct repeats are bound by
heterodimer.3 The third type of C6 ZFP has the ability to
bind to 2 zinc ions by six conserved cysteines, which are
critical in binding to DNA either in the form of monomer
or dimer.47
Due to the simplicity of using monomer in recognizing and binding to DNA, the type of C2H2 ZFP is commonly used in engineering ZFN. In particular, the most
ZFNs are engineered from the basic structure of naturallyoccurring mouse or human ZIF268. With regards to DNA
2376-3949/2015/1/003/013
doi:10.1166/gge.2015.1010
binding ability, Fairall et al. analyzed the crystal structure

of ZIF268 and identified the formation of hydrogen bonds
between the 1, + 2, + 3, + 6 amino acids in the -helix
of ZIF268 and the target DNA.8 The amino acids that are
involved in the formation of hydrogen bonds with the DNA
are theoretically responsible for the DNA sequence recognition. It was suggested that the 2nd amino acid is able
to recognize one base from the direction of 5 to 3 in a
single DNA strand, while the amino acids at 1, + 3, and
Tang et al.
+ 6 position recognize 3 bases from the direction of 3 to

5 in the other DNA strand.9 Thus, a single zinc finger
protein can recognize upto 4 nucleotides for physiological
regulation. However, from the technological point of view,
the string of 3 bases from 3 to 5 direction is easier to
handle in the zinc finger protein array. Thus, mutagenesis
experiments that focus on the 1+ 6 amino acids of the
ZFP -helix was performed to screen for the most efficient combination of amino acids for binding to a string
Li-Meng Tang was born in 1989. Currently, She is studying for her Masters degree
in pharmacology at the Institute of Pharmacy and Pharmacology, University of South
China, China. Her research interests are focused on gene diagnosis and the applications
of engineered nucleases (including ZFN, TALEN and CRISPR/Cas9) in gene modification and gene therapy.
Cui-Lan Zhou is a lecturer in the medical school at University of South China. Currently she is pursuing her Ph.D. studies in Central South University under the supervision of Professor Weijun Cai and Kai Li. She received her masters degree in molecular
pharmacology from University of South China in 2006. Her research areas of interests
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are molecular
diagnostic
and gene therapy.
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Zi-Fen Guo received her B.S.M from Hengyang Medical College, China. She has since
been teaching Pharmacology and Clinical Pharmacology Courses in the Department of
pharmacology at University of South China, and currently she is an Associate Professor
of pharmacology. Advised by Professor Duanfang Liao and Kai Li, she obtained her
Ph.D. degree in Pathology and Pathophysiology from University of South China in
2011. Her research focuses on molecular diagnostic and individualized medication.
Li Xiao is a Research Assistant in Molecular Medicine Center at the Second Affiliated

Hospital of Soochow University. Currently she is pursuing her Ph.D. studies in Soochow
University under the supervision of Professor Kai Li. She received her masters degree
in molecular pharmacology from University of South China in 2007. Her research areas
of interests are molecular diagnostic and gene therapy.
Gene Gene Edit. 1, 315, 2015
Tang et al.
Anderly C. Cheh received his BBiomedSc degree from the University of Melbourne
in Australia. During his undergraduate years from 2001 to 2002, he received significant
research training from apoptosis pioneers Professors David L. Vaux and David C. S.
Huang to study the mechanism of cell death at the Walter and Eliza Hall Institute for
Medical Research. Dr. Cheh subsequently move to the Murdoch Childrens Research
Institute to complete his Honours and Ph.D. studies on the genetic and epigenetic
regulation of centromeric chromatin between 2003 to 2008, under the supervision of
internationally renowned chromosome biologists Professor KH Andy Choo and Dr. Lee
H. Wong. From 2008 to 2013, he undertook his post-doctoral training with colorectal
cancer expert Associate Professor John Mariadason at the Ludwig Institute for Cancer
Research, where he investigated the genetic and epigenetic basis of colorectal tumour
syndromes and their response to novel targeted therapy. Dr. Cheh is currently a Research Fellow at The Walter and
Eliza Hall Institute of Medical Research and an Honorary Fellow at the University of Melbourne in Australia.
of 3 nucleotides. There are now engineered ZFPs available

the Fok I cleavage domain executes the nuclease activity
in recognizing all 3 string of GNN, most of ANN, some
when two reversed domains form a dimmer. Fok I is an
CNN, and a few TNN strings.1012 This strategy makes
endonuclease isolated from Flavobacterium okeanokoites.
the design and assembly of artificial ZFN easier and more
The recognition site of Fok I has an asymmetric recogpredictable. In addition to the genetically engineered ZFP
nition sequence of 5 -GGATG-3 : 5 -CATCC-3 . The cutbased on the backbone of ZIF268, other naturally occurting site is distinct to the recognition site and is 9 bp 3
ring ZFPs in the human genome have been successfully
downstream of GGATG and 13 bp upstream of CATCC.16
used in making ZFNs by Korean investigators.13 14
This feature of Fok I offer a unique opportunity of being
To be a practically useful endonuclease for gene editused as a genetically-engineered nuclease because it coning in mammalian genome, recognition of a string of 18
sists of two components of separate recognizing and cutbase pair in DNA sequence is the minimum requirement.2
ting domains. As compared with the 5 bp recognition site
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Therefore, at least 6 zinc finger
motifs are
needed toto:engiof wild
Fok I,de
engineered
nucleases usually have a
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neer a novel ZFN. Since a ZFN consists
of
two
monomers,
longer
recognition
sequence.
Thus,
the spacer between the
the minimal requirement of a monomer is a series of 3 zinc
2 recognition elements is a potential factor that may regfinger motifs. Typically, the 2 adjacent zinc finger motifs
ulate nuclease activity and should to be evaluated. For
are not directly linked to each other. Instead, a particuthis, Bibikova et al. investigated the contribution of spacer
lar string of amino acids such as the typical sequences of
length to the enzymatic activities of ZFNs.17 When the
TGEKP is commonly used between as the spacer between
separation between the 2 recognition elements was 4 bp,
2 zinc finger motifs.15 It was initially believed that the
the enzymatic activity was almost completely abolished.
zinc finger motifs may be serially linked to form a long
Increasing the length of the spacer sequence showed a
string without the losing their DNA binding ability. Howbimodal effect, with the highest enzymatic activity using
ever, through series of experimentation, we have now real8 bp spacer and the second highest activity contributed by
ized that the highest binding ability is only maintained in
a spacer of 16 bp.
strings containing 3 to 4 zinc finger motifs.
The development of ZFNs opens a new avenue for gene
The ZFN functions as a dimer, recognizing each singleediting with direct application in gene therapy. In constrand DNA sites by each of the monomer. The zinc fintrast to conventional gene knockout using spontaneous
ger motifs are responsible for the DNA recognition, while
homologous recombination, DNA repair mechanisms
Figure 1. Structure of Zinc finger nuclease. Each monomer is composed of ZFA and Fok I nuclease, it generally works when
two monomers form a dimer, F1F6 are zinc fingers, each zinc finger is connected through the linker. + 6, + 3, and 1 amino
acid of each zinc finger bind DNA.
Tang et al.
The major applications of gene editing using ZFNs can

be divided into 3 major categories:
(1) disease modeling at cellular level or intact organismal
level;
(2) genome modification of economically interested
species for agriculture and industry; and
(3) human gene therapy. A short summary of these examples is listed in the Table I.
Disease Modeling at Cellular Level or Intact
Organismal Level
ZFNs mediated gene editing has been widely used in the
modification of many types of cells, including leukemia
cell line K562,33 induced pluripotent stem cell (iPS),34 and
Figure 2. Two cell repair mechanisms: NHEJ and HR.
embryonic stem cells.35 Genetic engineering approaches
such as traditional gene knockout and transgenic techare greatly activated following double-stranded breaks
nologies and the latest ZFN-associated gene editing are
induced by ZFNs. Subsequent to the formation of
extremely powerful in the development of animal moddouble-stranded breaks, both non-homologous end-joining
els for human diseases. Conventional technologies based
(NHEJ) and homologous-directed repair (HR) machineron spontaneous homogenous or heterogeneous recombinaies are recruited for genomic repair (Fig. 2). NHEJ is
tion have led the production of a suite of useful genetia DNA template independent mechanism by which a
cally modified animals. In medical research, both mouse
series of proteins act to join the break ends through ligand rat disease models are frequently used tools. Howation. In contrast, HR is DNA template dependent mechever, the difficulty in obtaining rat embryonic stem (ES)
anism that allows for the integration of external DNA
cells restrains the development of rat disease models via
fragments into the host genome.18 It was reported that
traditional methods. In 2009, Geurts et al.29 reported a
the HR efficiency increases up to several thousand fold
successful
gene knockout
in rat by injecting ZFN into ferDelivered by Ingenta to: Universidad
de Santiago
de Chile
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breaks inducedOn:
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egg,
which
paved
a new way for the production
ZFN.19
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Publishers
of genetically
engineered animals. DNA microinjection of
The high efficiency of gene editing after the producfertilized eggs, coupled with ZFN technology, provides a
tion of double-stranded breaks open up the opportunity to
possible route to construct genetically-modified models for
several kinds of gene editing, including both gene knockthe species by which ES cells cannot be obtained, which
in and gene knock-out. Gene knock-in can be used to
also greatly shorten the time required for experimentation.
fix abnormal or mutated gene and to introduce external gene pieces such as reporter genes by homologous
Genome Modification of Organisms for
recombination.2022 By comparison, NHEJ is a template
Agriculture and Industry
independent mechanism that is more efficient than homolThere have been multiple recent successes in modifyogous recombination. Depending on the gene target to be
ing the genome of organisms for agriculutre and indusedited, NHEJ can work to either activate or inactivate an
trial uses. Shukla et al.36 has successfully employed the
internal gene of interests.
ZFN technology to produce a herbicide-resistant and
phytate levels-reduced, double-advantage corn, while
APPLICATIONS AND THE CURRENT
Townsend et al.28 has produced a herbicide-resistant
LIMITATION OF ZFNS
tobacco plant using similar approach. These 2 landmark
studies, published in Nature, highlighted the great potential
ZFN is the first engineered endonuclease to be used for
of using ZFN technology in producing transgenic plants
gene editing. In the past few years, ZFNs have been widely
for agriculture. Currently, genetical-engineering domesused in gene editing of specific targets for gene knockticated animals are still a challenge. Galli et al.37 has
out, gene integration, and gene replacement. The latter two
recently employed the microinjection ZFN and nuclear
can be broadly considered as gene knock-ins. As one of
transfer technology to successfully cloned pigs with EGFP
the pioneer application, the yellow gene was successfully
sequences.38 This provides a simple and efficient way forknock-outed in Drosophila by ZFN via NHEJ.20 There are
ward for gene editing in livestock. As pigs and other
an ever-increasing number of model organisms in which
animals can be readily used for organ transplantation,
genes are edited by ZFNs or other similar types of engiZFN technology may be utilized for cloning geneticallyneered endonucleases. As tabulated in Table I, some of the
modified pig with reduced immune response-a future applispecies including Drosophila, zebrafish, C. elegans, mouse,
cation that can be considered for organ transplantation.
rat, and pig.2332
6
Tang et al.
Table I.
Applications of ZFNs in different species.
Applications
Disease modeling
at cellular level
or intact animal level
Species
Gene
Drosophila melanogaster
Yellow
Rosy
Brown
slc24a5 (golden)
ntl
kdrl (kdr, flk)
Synthetic QQR target
Mdr1a
Jag1
Notch3
Il2rg
Rab38
IgM
ADH1
TT4
SuRA
SuRB
Ipk1
Zp15
Exogenous eGFP
CCR5
CXCR4
IL2RG
F9
Ush1c
-synuclein
AAVS1
Danio rerio
Caenorhabditis elegans
Mouse
Rat
Genome modification of
economically interested
species for agriculture
and industry
Arabidopsis
Nicotiana tabacum
Zea mays
Human gene therapy
pig
Homo sapiens
Modification methods
NHEJ
NHEJ, HR-mutation
NHEJ
NHEJ
NHEJ
NHEJ
NHEJ, HR
NHEJ
NHEJ
NHEJ
NHEJ
NHEJ
NHEJ
NHEJ
NHEJ
NHEJ
NHEJ, HR-mutation
NHEJ, HR-insertion
NHEJ, HR-insertion
unknown
NHEJ
NHEJ
HR-insertion
HR-insertion
HR-mutation
HR-mutation
NHEJ, HR-insertion
References
[20]
[23]
[23]
[24]
[24]
[25]
[27]
[31]
[31]
[31]
[30]
[29]
[29]
[26]
[26]
[28]
[28]
[36]
[36]
[32]
[39]
[42]
[46]
[47]
[48]
[49]
[53]
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Human Gene Therapy
therapy
to cure AIDS/HIV will no longer be a
Copyright:
dream. Publishers
The use of ZFN technology to generate
knockout American
and gene Scientific
In addition to AIDS, hepatitis B is another human disreplacement could potentially be a very promising methodease that has been targeted by novel ZFN technology.
ology for gene therapy, if under the premise of possibly
Using designer ZFN targetting to hepatitis B virus, Cradick
reducing the toxicity. Investigations on ZFN-related gene
et al.45 performed proof-of-principle experiments demontherapy are currently underway and can be divided into 2
strating the specific killing of hepatitis B virus to certain
research trends: gene knockout and gene repair. The develextent, which provides a new avenue for the treatment of
opment of treatment options of HIV using ZFN technology
hepatitis B and other similar diseases.
belongs to gene knockout studies. HIV enters the body
The repair of dysfunctional genes for gene correction
via the CCR5 receptor. Deleting a segment of 32 bp in
using ZFN is largely limited to single gene disorders. The
the CCR5 gene can make T cells exhibit anti-HIV activintroduction of ZFN generates DSB and cellular repair
ities, and the use of ZFN technology can theoretically be
mechanisms using homologous recombination can theoemployed to edit the CCR5 gene in T cells to provide
retically serve the purpose of repairing dysfective genes.
potential cure for AIDS. Number in vitro studies have now
Scientists have now successfully applied this method for
been performed and demonstrated that this approach is
in vitro gene correction of a variety of human disquite promising.3941 Yuan et al.42 has recently reported
eases, including X-linked severe combined immunodefithat in addition to modifying CCR5, knockout of CXCR4
ciency disease,46 hemophilia B,47 Usher syndrome48 and
gene can make mouse cells resistant to HIV. By com43
so on. Soldner et al.49 was the first group to modify a
parison, Didigu et al. generated a double knockout of
single disease-causing mutation in human stem cells with
CCR5 and CXCR4 cell line using 2 designer ZFNs and
ZFN without causing changes any other part of the stem
achieved a significant enhancement of resistance to HIV
cell genome. The researchers carefully inserted or removed
infection. Holt et al.41 has recently provide compelling evia single base pair in the Parkinsons disease-causing
dence supporting the notions that HIV can be clinically
-synuclein (alpha-synuclein) gene in induced pluripotent
cured by ZFN transformed autologous hematopoietic stem
stem (iPS) cells. This approach have the ablity to correct
cells.44 Excitingly, ZFN-based drugs developed by Sangfor point mutations that were present in the patients iPS,
amo for the treatment of AIDS has recently entered the
and may be readily applicable for the development of treatPhase 1 clinical trial in humans. Based on these proof-ofment strategies for other single gene disorders. For genetic
principle studies, it is believed that in the near future, the
diseases that are caused by the constitutional deletion of a

large DNA fragment such as -thalassemia, it is difficult
to use ZFN for gene repair via the HR mechanism. As
lentivirus-mediated gene transfer may cause cytotoxicity,
many recent studies focus on integration via the AAVS1
gene locus,5052 AAVS1 is a locus in PPP1R12C gene on
human chromosome 19. It was recently demonstrated that
the DNA can be faithfully integrated into this site without affect expression of the surounding genes,53 hence the
cytotoxicity is minimized via this approach. Therefore,
AAVS1 locus is potentially a good target site for ZFNbased treatment of genetic diseases.
Tang et al.
cloning methods. Pruett-Miller et al. compared Bacterial

2-Hybrid (B2H) selection-based method with a pair made
using modular-assembly, which results in better output,
more efficient and less toxicity when compared with the
B2H strategy.54 Third, proper Fok I domain need to be
chosen and assembled to ZFN. Typically, wild-type Fok I
cleaving domain was used in this process. As ZFP homodimer showed a higher off-target effect than that of the
heterodimer, the recent trends have been using genetically modified Fok I domain for the development of ZFN.
Depending on the application (gene repair, knock-in or
knock-out), eitherhomologous recombination (HR) or nonhomologous ending joining (NHEJ) will be selected as the
mechanism of gene editing by the biological system.
Current Limitations of ZFNs

Despite the success in the use of ZFN technology in gene
Optimization of the Zinc Finger Binding Domain
editing, there are also many obstacles that severely limited
Zinc finger arrays assembled from single zinc finger with
its use for wider application. Firstly, the selection of tarhigh affinity does not necessarily have high affinity as
get gene sequences for ZFN binding with high specificity
a whole. Zinc finger binding domain constructs exert
and affinity is a major challenge with the target sequence
context-dependent cellular effects, therefore engineered
3 triple each combination of sub-assembled zinc finger but
zinc fingers must be filtered after the assembly for the
did not specifically identify the sequence 9 bp, which adds
selection of zinc finger bind domain with the strongest
great difficulty in ZFP screening and assemblying.
affinity to target DNA. There are currently 4 frequently
Furthermore, how to select zinc finger frame, the numused assembling method in the literature including Modber of zinc finger and the polypeptides connected ZFP
ule Assembly (MA), OPEN (Oligomerized pool engizinc fingers to optimize the ZNFs specificity and affinity
neering) scheme,55 Wolfes scheme,24 a context-dependent
must be considered. Using experience to infer the ZFNs
assembly (CoDA) scheme.56 MA program is simple, fast
effect is undesirable, because its effects may vary greatly
Santiago
and isdenot
limited de
to Chile
zinc finger DNA sequences and
in different cells or different species.
Sangamos monopoly
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13 23:21:29
framework.
However
it was recently reported a very
severely limits the widespread application
of ZFNAmerican
technol- Scientific Publishers
Copyright:
low
probability
(630%)
of success to generate active
ogy. ZFN off-target problem somewhat limited its appli57 58
OPEN
program
seems to be able to generate
ZFN.
cation, cytotoxicity caused by non-specific cleavage may
much
higher
success
than
the
MA in ZFN development,
cause great harm in cells, embryos or individuals, espebut
the
OPEN
program
is
complex,
time consuming, and is
cially in gene therapy. A large number of studies have
limited
to
all
GNN
and
a
small
part
of TNN. For example,
shown that, when the DSB in the genome of the cells
if
zinc
finger
array
is
composed
of
3
zinc fingers, the proboccurs, cells tend to use NHEJ repair mechanism but not

-GNNGNNGNN-3
is
less
than 4%, as a result
ability
of
5
HR mechanism, which increases the cytotoxicity while not
the
DNA
sequence
that
can
be
identified
by the two left
serve the purpose of repair gene replacement. And when
and right arrays is as low as 0.16%,59 thus OPEN program
using ZFN technology in the clinical setting, whether ZFN
can only configure 3 zinc finger arrays. By comparison
injection causes the body to produce an immune response,
to OPEN, Wolfes program has the same advantages and
as well as on the human body in addition to the purpose of
disadvantages, but the process development using Wolfes
treatment will cause any side effects? We must face these
program is much simpler.60 Similarly to other programs,
ZFN application problems. So in recent years, to solve the
CoDA
scheme also perform direct assemblely of ZFPs,
above problems, the scientists have done a lot of research
but
due
to the fact that CoDAs zinc finger design is
to improve ZFN technology.
based on the OPEN program, it has significant limitations. Each of the four programs have their own advanADVANCES IN ZFN CLONING
tages and limitations. When the target sequence is mostly
Gene editing using ZFN is a multiple-step process.
GNN, OPEN, Wolfes, and the CoDA programs will be
In order to obtain efficient gene editing, optimal sequences
the ideal platform of choice for ZFN development because
first need to be selected based on surrounding sequences
the technology is more mature and processes are effiof the target genes of interests and then a list of the corcient. However, due to the complexity and time-consuming
responding ZFP arrays are cloned. Second, DNA bindnature of using OPEN and other related methodologies,
ing ability of the zinc finger binding domains need to be
in recent years, many researchers have focused on the
tested before assembling into ZFN as the DNA binding
improvement of the module assembly program. As early
ability is crucial for possessing efficient enzymatic activas 2001, Isalan et al.61 developed a program different
from the 4 above-mentioned ZFA construction scheme or
ities. Different affinity assays can be used for different
8
Tang et al.
programs. This newly developed method is based on 2 preZFP. The usefulness of this improvement was confirmed
prepared zinc finger phage display libraries #12 and #23.
in many recent studies.65 66 Despite this, the correlation
between increase activity/specificity and ZFP number is
Separately generated highaffinity two-module (2F) ZFA
not linear, this is because that a few ZFN decreases effifrom these libraries were subsequently assemble into a 3
ciency when the number of zinc finger increases. Based on
finger Zinc finger arrays. It may seem somewhat similar
the foundation of these principles, the authors developed
to CoDA, but CoDA has only 1 common F2 zinc fina B-score scoring system that facilitates the wide-use of
ger. The principle of this method is as follows: The zinc
the direct module assembly method. However, the scorfinger in libraries #12 identify 3 -HIJKLMGGCG-5 , the
ing system is far from perfect and significant improvement
zinc finger in library #23 identify 3 -GCGGMNOPQ-5 as
needs to be performed in order for obtain precise predicthe target sequence HIJKLM and MNOPQ in the target
tion of ZFN activity with pre-screening in the near future.
sequences are random nucleotides and shared 1 in base
By altering ZFNs intrinsic backbone, ZFN techniques
pair common designated as M. Based on the zinc fincan also be improved. ZFNs are commonly and classigers generated from the 2 libraries, the M base pair of
cally build from ZIF268 backbone skeleton. Recently Bae
the zinc finger nucleotide are removed and subsequently
et al.13 screened 56 kinds of natural zinc finger encoded
synthesized into the 3F zinc finger arrays, which now conby the human genome and used them directly to contains 9 bp with the sequence of HIJKLMNOPQ. This
struct ZFN. As a validation study, Kim et al.14 used these
methodology is not only simple, but more importantly, it
natural zinc fingers to construct ZFN and demonstrated
makes the sequence recognition of zinc finger no longer
62
enhanced activities when compared with one derived from
restricted to any triplet codons. Gupta et al. developed
the ZIF268 skeleton. Thus, naturally occuring zinc fingers
a ZFN construction scheme named finger stitching, and
63
of the human genome are perhaps the better backbone of
performed a full validation study. The method is based
choice for constructing ZFNs.
on 2F-MA, which almost exclusively recognize the NG
The linker sequences connecting between the invididual
junction of GNN-GNN. Finger stitching can identify 2
zinc
fingers also have an impact on ZFN activity and specizinc finger random joints of the 2F module. The recognificity.
Classical linker sequence between the zinc fingers is
tion sequence is GRN-NYG, wherein R represents A or
TGEKP.
It was recently reported that the additional of SerG and Y represents C or T. This method can efficiently
ine
to
the
TGEKP (i.e., TGSQKP) sequence significantly
identify zinc finger DNA sequences with 3-fold increase
de Santiago
de of
Chile
increases
the activity
the engineered ZFN.62 67 Howin the probability of successful design,
and is not limited
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ever, further validation experiments are required to confirm
the direct
mod- Scientific
to 3F ZFA. 2013, Bhakta et al.64 extended
Copyright:
American
Publishers
whether ZFNs activity can be enhanced by increasing the
ule assembly method, where they use a drop-out linker
length of the linker as there is one study presents the conmethod (Fig. 3) to study the activity of ZFAs configured
trary results.64 Wilson et al.68 have recently studied how
in a form of 36 zinc fingers. The result of this improved
the different types of linker length and spacers affects the
method is a significant increase in ZFN activity and speciZFN activity. They found 2 to 4 amino acid linker have the
ficity as correlated with the increase of the number of
highest efficiency of the 5 to 6 bp spacer sequence, while
5 amino acids linker length have the highest efficiency of
the 6 to 7 bp spacer sequence. Similarly, Anand et al.69
have recently demonstrated that an increase in the linker
sequence between two zinc fingers makes the 2 zinc fingers skip a maximum length of 10 bp sequence. Together,
these results highlighted the importance of amino acid
linker in regulating ZFN activities and specificities.
The construction of ZFN can also be produced from a
number of new methods. Latest research70 indicates that
a ZFN monomer and TALEN (transcription activator like
effector nuclease) monomer can form a hybrid nucleic acid
enzyme, which enhanced specificity in the performance
of the different targets. These hybrids can be produced in
different cell types, including human induced pluripotent
stem (iPS). Yusuke et al.71 invented a sandwiched ZFN
(Fig. 4), composed of 2 ZFN domains and a sandwiched
Staphylococcus aureus nuclease (SNase). This novel ZFNenzyme can cut DNA in the form of monomers. It is
Figure 3. Drop-out linker. Hind III, Bsm I etc. are the restricreported that the advantages of this enzyme is its ability
tion enzyme sites, firstly synthesis of a 6F zinc finger arrays
indistinguishing DNA substrate cleavage products and to
then excised by restriction enzyme to generate 5F, 4F, 3F zinc
finger arrays.
improve the efficiency of ZFN. In addition, Fujii et al.72
Figure 4. Sandwiched ZFN. SNase is a Sandwiched single

Staphylococcus aureus nuclease, the zigzag line is the linked
polypeptide.
Tang et al.
a high active ZFN monomer could assemble a new ZFN

that can produce biological activities,74 in order to save
these monomers of low activity, Zhang et al.75 was the
first group to invent a reporting system for high sensitivity
detection of low activity ZFN. They assembled ZFN from
highly active monomers and monomers with low activities. They demonstrated that the engineered ZFNs derived
from this reporting systems function properly and that the
approach is economical. This new method also produces
lower ZFN activity and reduces the cytotoxicity.76
have invented a new construct method of ZFN, named

OLTA (OverLap extension PCR and TA-cloning), in which
the zinc finger DNA encoding fragment was synthesized
by PCR. A short sequence primer homologous to the DNA
recognition helix domain is responsible binding to ZFP,
while different primers encodes different recognition helix
sequences. The 46 zinc fingers were synthesized by overlap extension PCR and cloned into a vector with nuclease
using TA cloning. The advantage of this new approach is
that to the making of the construct of ZFN is easy, quick
and cheap, however the drawback is obvious. If not perform properly, PCR often results in high base mismatch,
and that a single base mismatch could results in detrimental effects in the activity of ZFN.
Modification of the Cleaving Domain of Fok I

In recent years, there have been a number of studies
that investigate on effects of modifying the Fok I cleavage domain to improve ZFNs specificity. DNA shearing requires 2 Fok I units for dimerization and at least
one to bind DNA. Dimerization is a process independent of DNA shearing. Heterodimers, homodimers and
monomers of Fok I all exhibit DNA shearing activities, with different recognition sequences and non-specific
behavior that may results in increases in ZFN toxicity.77
To improve ZFN specificity and reduce the toxicity, dimerization region of Fok I is commonly modified to reduce
their homo-dimerization activity. ZFN pair constructed
from two variants, only the hetero-dimerization induces
specific cleavage, while homo-dimerization induces low
Optimization of the Reporting and
cleavage activity. Following this principle, Sangamo synDelivered by Ingenta to: Universidad
de aSantiago
de Chile
Screening System
thesised
mutant Fok
I with two variants, Q486E: I499L
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In addition to direct module assembly method, OPEN,
and
E490K:
I538K
(also
called EL_KK), each having 10Copyright: American Scientific Publishers
CoDA and other methods all required to pre-screening for
fold reduction in cytotoxicity when compared to the wildZFN development. Various bioinformatic programs have
type Fok I. In 2007, Szczepek et al.78 synthesised a mutant
been developed for screening and testing for newly derived
Fok I, in which the residue pair of the dimerization region
ZFN. Typically, a ZFA library is first built, then merged
was mutated by site-direct mutagenesis. The two variants
into a transcriptional activation domain to form a zinc
R487D and D483R (also named DD and RR) are now
finger-activating factor (also abbreviated as ZFA). Subsecommonly used in ZFN technology. Similarly, Cathomens
quent to this, a reporter gene expression system carrying
laboratory generated a pair of variants having 2 Fok I
the target site is used to detect the intensity of the reporter
mutants D483R: I538V and R487D: I499A (also called
gene expression to filter select highly active ZFAs. TradiRV_DA). These mutants have reduced some cytotoxictional reporting and screening systems are Phage display,
ity when compared to wild-type. Based on the work of
yeast two-hybrid (Y2H), bacterial two-hybrid (B2H), bacSangamo and Szczepek, Ramalingam et al.79 synthesised
terial one-hybrid (B1H), and so on. However, these screen2 additional variants, REL_DKK and RELV_DKAK, and
ing systems are laborious and time-consuming, therefore
a new variant, the Fok I_Sts I. The principle behind the
researchers have focused on developing new screening sysdesign was the use of partial sequence of Sts I restriction
tem to improve ZFN technology. Wang et al.73 invented a
enzymes to replace dimerization domain of Fok I. In these
novel system, which can simultaneously screen and valnew variants, when REL_DKK was assembled with a
idate ZFN activity. They used yeast AH109 to directly
ZFA of 34 fingers, the authors demonstrated a remarkobtain active ZFNs. The system is based on OPEN and
able ability to reduce toxicity in human cells. FurtherGal4 reporting system, with a special reporter plasmid conmore, Guo et al.65 used directed evolution approach at a
taining a 30-bp Gal4 homologous arm and a ZFN target.
dimerization region of Fok I to generate a hyper-activation
ZFN binds to the target and leads to DSB, which is subsevariant, Sharkey. Compared with conventional techniques,
quently repaired by single-strand annealing (SSA) mechaSharkey exhibited 15 times higher ZFN cleavage activnism. Although based on OPEN, this system can produce
ity, and is compatible with a variety of old and new ZFN
up to 24-bp specific recognition of ZFN and is capable of
skeletons.80 In 2012, Schierling et al.81 made a simple
generating active ZFN or verify the constructed ZFN activchange of the Fok I endonuclease to PvuII and built a new
ity in a time efficient manner. Reported that a low-active
ZFN. Direct experiments performed the authors proved
that ZF-PvuII cleavage of the target is 1000 fold more
ZFN monomer which can not produce ZFN activity and
10
Tang et al.
efficient than cleavage for the non-target sites. Importantly,

ZF-PvuII treatment does not produce any excess enzyme
activities on the substrate, nor produced any non-specific
cleavage in long time culture. This novel ZFN variant may
be a valid alternative to existing ZFNs.
required to exposure the cells to ZFNs (and therefore to

minize off-target effects), but also avoid the cells to be
exposed to any other genetic material that is not part of
the knockin gene. In addition, it is was recently reported
that directly transfering the ZFN-encoding DNA fragment
in the cells, rather than using plasmids as the vector,
avoids off-target homologous recombination and reduce
Optimization of ZFN Modification Technology
cytotoxicity.90
NHEJ and HR are the 2 major mechanisms by which ZFN
In addition to modifying the ZFN itself, optimization
introduce gene editing. As previously mentioned, NHEJ
for DNA homology of the donor sequence is also imporis the more favorable mechanism over HR in cell. This
tant. The donor sequence usually need to be about 750 bp
processes a problem as NHEJ is more prone to introduce
on each arm to generate HR. Salvatore et al.83 provides
novel mutations. So, how to avoid NHEJ mechanism at
a new donor, only about 50 bp in length. When the
the time of HR has become the central in the improve82
donor sequence was designed to contain a single-stranded
ment of ZFN technology. Ramirez et al. introduced a new
5 protruding that is complementary with ZFN notch,
modification method of ZFN, through inactivating Fok I
the rate of integration of the exogenous genes can be
restriction enzyme activity in a monomer of a ZFN dimer
greatly enhanced. This is because the mechanisms of cell
to generate ZFN cutout (ZFNickases), and then produce
are using NHEJ instead of HR and this approach simDNA single-stranded break (SSB) in the target sequence.
plifies the design of the donor DNA. It has also been
SSB incision may induce HR but results in a lower frerecently reported91 92 that single-stranded oligonucleotide
quency than conventional DSB approach. The advantage is
(ssODN) can be used as an alternative to traditional
that it can avoid NHEJ, thus greatly reduce the degree of
double-stranded donor DNA, These studies demonstrated
cytotoxicity. Salvatore et al.83 used 2 ZFNs simultaneously
that ssODN improves ZFN activitiy and increases the freto cut the target sequence to generate 2 DSBs, and then
quency of HR mechanisms within the cell.
integrated the donor DNA sequence by NHEJ mechanisms.
Interestingly, the biologic activity of ZFNs can be modThis methodology led to approximately 50% of successified by using a variety of small molecules as well.
ful deletion events for the inserted exogenous gene. This
DNA end-processing enzyme was reported to increase
method hase allowed the improvement in efficiency for the
by in
Ingenta
to: Universidad
de Santiago
Chileelicited by ZFNs.93 Furtherthe gene
disruptionde
effects
ZFN technology, but it may Delivered
also increase
cytotoxicity.
IP: 158.170.255.25 On: Fri,more,
22 Aprthe
2016
23:21:29
addition of proteasome inhibitors such as
MG132 can increase the stability of ZFNs in gene editOptimization of Cell Transfection Technology
ing experiments.96 Other interventions such as increasing
Typically, the gene encoding ZFN is introduced into the
the transduction efficiency and changing the cell culture
cell via DNA plasmid, viral vectors, or in vitro transcrip84
conditions may also enhance the gene editing effects.97 98
tion of the mRNA. However, the reagents that are used
Non-toxic cell cycle modulator Indirubin was reported in
to transfect DNA plasmid or the mRNA by electricical
augmenting AAV transduction.97 In addition, positively
or liposomal transfer method may be toxic to the cell,
gene-edited cells can be significantly enriched by magnetic
depending of the cell type. This is a major limitation of the
85
separation, co-expression of florescence reporter gene, or
ZFN technology that needs to be further optimized. The
selection using antibiotic resistant gene.94 95
use of viral vectors is also another limiting factor because
they are difficult to assemble and could cause potential
Off-Target Effects of ZFN
immunogenicity issues. Recently, adeno-associated virus
Despite much success in the development of ZFN technol(AAV) has been proven to be safe in humans and does
ogy, off-target effects may contribute to genetic toxicity
not produce major side effects as demonstrated by multiand side effects when using ZFNs to study the normal
ple clinical trials. AAV is a promising ZFN vector. They
physiology of cells and organisms. To circumvent this
have already been used to enhance HR and drive ZFNproblem, two strategies can be employed to minimize
mediated gene correction in vivo.86 Efficient AAV assempotential off-target effects. The first strategy is to perform
bly occurs only in expression cassette that is less than 4.2
in vitro selection using a partly degraded library (based
kb; however the insert size is large enough to accommoon the expected ZFN cut sites).99 The second option is
date 2 ZFN monomers and an engineered construct donor.
to identify possible off-targets at the genomic level based
It has been reported that the use of AAV scheme demonon IDLV DNAs.100 However, due to the limitations on
strated to be feasible and improves the ZFN technology.87
the sensitivity of the proposed methodologies described
Integration-defective lentiviral vector is (IDLV) is another
above, many off-targets may still be missed.101 As a result,
attractive alternative. ZFNs can be transfected into the
88
Sander et al. suggested that a unbiased bioinformatics analmultiple tolerated cell types. Recently, there is a report
on a new form of transfection, where purified ZFN proysis should be applied to recover wider spectrum of the
teins can directly introduced into the cell to cause a genetic
off-targets, using specific programs such as ZiFiT,102 Zinc
89
modification. This method not only by shorten the time
Finer Tools,103 and ZFN-Site.104
11
Tang et al.
In addition to the in silico analysis, novel experimental

approaches are urgently needed to screen for genome-wide
off-target effects. Both beta-Gal-based assays and the use
of antibiotic resistance genes have been widely performed
in evaluation of the enzymatic activity of engineered nucleases. They are expected to be useful in the development of
more sensitive technologies as screen for off-target effects
by further optimization of there reporter genes or some
other new reporter system.
the construction of artificial transcription factors. Nat. Biotechnol.

21, 27580.
13. Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., and Kim, J. S. (2009).
Targeted genome editing in human cells with zinc finger nucleases
constructed via modular assembly. Genome Res. 19, 127988.
14. Peisach, E. and Pabo, C. O. (2003). Constraints for zinc finger
linker design as inferred from X-ray crystal structure of tandem
Zif268-DNA complexes. J. Mol. Biol. 330, 17.
15. Looney, M. C., Moran, L. S., Jack, W. E., Feehery, G. R.,
Benner, J. S., Slatko, B. E., and Wilson, G. G. (1989). Nucleotide
sequence of the FokI restriction-modification system: Separate
strand-specificity domains in the methyltransferase. Gene 80,
Acknowledgments: We acknowledge the financial sup193208.
16. Bibikova, M., Carroll, D., Segal, D. J., Trautman, J. K., Smith, J.,
ports of the Chinese National 863 Major Grant (No.
Kim, Y. G., and Chandrasegaran, S. (2001). Stimulation of homol2012AA020905), the National Natural Science Foundation
ogous recombination through targeted cleavage by chimeric nucleof China (No. 81301267), the Priority Academic Program
ases. Mol. Cell. Biol. 21, 28997.
Development of Jiangsu Higher Education Institutions, the
17. Capecchi, M. R. (1989). Altering the genome by homologous
recombination. Science 244, 128892.
Innovation Project of Jiangsu Graduate Education (No.
18. Klug, A. (2010). The discovery of zinc fingers and their appliCXZZ11_0117) and Jiangsu Provinces clinical medical
cations in gene regulation and genome manipulation. Annu. Rev.
science and technology program (No. BL2013016).
Biochem. 79, 21331.
19. Bibikova, M., Golic, M., Golic, K. G., and Carroll, D. (2002). Targeted chromosomal cleavage and mutagenesis in Drosophila using
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