Zinc nger nuclease

Zinc-nger nucleases (ZFNs) are articial restriction

enzymes generated by fusing a zinc nger DNA-binding
domain to a DNA-cleavage domain. Zinc nger domains
can be engineered to target desired DNA sequences and
this enables zinc-nger nucleases to target unique sequences within complex genomes. By taking advantage
of endogenous DNA repair machinery, these reagents can
be used to precisely alter the genomes of higher organisms.

with enhanced cleavage activity that the authors dubbed

Sharkey.[5] Structure-based design has also been employed to improve the cleavage specicity of FokI by
modifying the dimerization interface so that only the intended heterodimeric species are active.[6][7][8][9]

2 DNA-binding domain
The DNA-binding domains of individual ZFNs typically
contain between three and six individual zinc nger repeats and can each recognize between 9 and 18 basepairs.
If the zinc nger domains are perfectly specic for their
intended target site then even a pair of 3-nger ZFNs that
recognize a total of 18 basepairs can, in theory, target a
single locus in a mammalian genome.

DNA-cleavage domain

Various strategies have been developed to engineer

Cys2 His2 zinc ngers to bind desired sequences.[10]
These include both modular assembly and selection
strategies that employ either phage display or cellular selection systems.
The most straightforward method to generate new zincnger arrays is to combine smaller zinc-nger modules
of known specicity. The most common modular assembly process involves combining three separate zinc ngers
that can each recognize a 3 basepair DNA sequence to
generate a 3-nger array that can recognize a 9 basepair
target site. Other procedures can utilize either 1-nger or
2-nger modules to generate zinc-nger arrays with six
or more individual zinc ngers. The main drawback with
this procedure is the specicities of individual zinc ngers can overlap and can depend on the context of the
surrounding zinc ngers and DNA. Without methods to
account for this context dependence, the standard modular assembly procedure often fails unless it is used to
recognize sequences of the form (GNN)N.[11]

A pair of two ZFNs with three zinc ngers each are shown introducing a double-strand break. Subsequent to this, the double
strand break is being repaired through either homologous recombination or non-homologous end joining.[1]

The non-specic cleavage domain from the type IIs

restriction endonuclease FokI is typically used as the
cleavage domain in ZFNs.[2] This cleavage domain must
dimerize in order to cleave DNA[3] and thus a pair
of ZFNs are required to target non-palindromic DNA
sites. Standard ZFNs fuse the cleavage domain to the Cterminus of each zinc nger domain. In order to allow the
two cleavage domains to dimerize and cleave DNA, the
two individual ZFNs must bind opposite strands of DNA
with their C-termini a certain distance apart. The most
commonly used linker sequences between the zinc nger
domain and the cleavage domain requires the 5' edge of
each binding site to be separated by 5 to 7 bp.[4]

Numerous selection methods have been used to generate

zinc-nger arrays capable of targeting desired sequences.
Initial selection eorts utilized phage display to select
proteins that bound a given DNA target from a large
pool of partially randomized zinc-nger arrays. More recent eorts have utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian
cells. A promising new method to select novel zinc-nger
arrays utilizes a bacterial two-hybrid system and has been
dubbed OPEN by its creators.[12] This system combines
pre-selected pools of individual zinc ngers that were
each selected to bind a given triplet and then utilizes a sec-

Several dierent protein engineering techniques have

been employed to improve both the activity and specicity of the nuclease domain used in ZFNs. Directed
evolution has been employed to generate a FokI variant
1

3 APPLICATIONS

ond round of selection to obtain 3-nger arrays capable

of binding a desired 9-bp sequence. This system was developed by the Zinc-Finger Consortium as an alternative
to commercial sources of engineered zinc-nger arrays.

tinct DNA sequences. The ZFN-encoding mRNA was

injected into one-cell embryos and a high percentage of
animals carried the desired mutations and phenotypes.
Their research work demonstrated that ZFNs can specif(see: Zinc nger chimera for more info on zinc nger ically and eciently create heritable mutant alleles at loci
of interest in the germ line, and ZFN-induced alleles can
selection techniques)
be propagated in subsequent generations.

Applications

Similar research of using ZFNs to create specic mutations in zebrash embryo has also been carried out by
other research groups. The kdr gene in zebra sh encodes for the vascular endothelial growth factor-2 receptor. Mutagenic lesions at this target site was induced using ZFN technique by a group of researchers in US. They
suggested that the ZFN technique allows straightforward
generation of a targeted allelic series of mutants; it does
not rely on the existence of species-specic embryonic
stem cell lines and is applicable to other vertebrates, especially those whose embryos are easily available; nally, it
is also feasible to achieve targeted knock-ins in zebrash,
therefore it is possible to create human disease models
that are heretofore inaccessible.

Zinc nger nucleases have become useful reagents for

manipulating the genomes of many plants and animals
including arabidopsis,[13][14] tobacco,[15][16] soybean,[17]
corn,[18] Drosophila melanogaster,[19] C. elegans,[20] sea
urchin,[21] silkworm,[22] zebrash,[23] frogs,[24] mice,[25]
rats,[26] rabbits,[27] pigs,[28] cattle,[29] and various types
of mammalian cells.[30] Zinc nger nucleases have also
been used in a mouse model of haemophilia[31] and an
ongoing clinical trial is evaluating Zinc nger nucleases
that disrupt the CCR5 gene in CD4+ human T-cells as
a potential treatment for HIV/AIDS. ZFNs are also used
for the creation of a new generation of genetic disease
models called isogenic human disease models.
3.2

3.1

Disabling an allele

ZFNs can be used to disable dominant mutations in heterozygous individuals by producing double-strand breaks
(DSBs) in the DNA (see Genetic recombination) in the
mutant allele, which will, in the absence of a homologous
template, be repaired by non-homologous end-joining
(NHEJ). NHEJ repairs DSBs by joining the two ends together and usually produces no mutations, provided that
the cut is clean and uncomplicated. In some instances,
however, the repair will be imperfect, resulting in deletion
or insertion of base-pairs, producing frame-shift and preventing the production of the harmful protein.[32] Multiple pairs of ZFNs can also be used to completely remove entire large segments of genomic sequence.[33] To
monitor the editing activity, a PCR of the target area
will amplify both alleles and, if one contains an insertion, deletion, or mutation, it will result in a heterduplex
single-strand bubble that cleavage assays can easily detect. ZFNs have also been used to modify disease-causing
alleles in triplet repeat disorders. Expanded CAG/CTG
repeat tracts are the genetic basis for more than a dozen
inherited neurological disorders including Huntingtons
disease, myotonic dystrophy, and several spinocerebellar ataxias. It has been demonstrated in human cells that
ZFNs can direct double-strand breaks (DSBs) to CAG repeats and shrink the repeat from long pathological lengths
to short, less toxic lengths.[34]
Recently, a group of researchers have successfully applied the ZFN technology to genetically modify the gol
pigment gene and the ntl gene in zebrash embryo. Specic zinc-nger motifs were engineered to recognize dis-

Allele editing

ZFNs are also used to rewrite the sequence of an allele by

invoking the homologous recombination (HR) machinery to repair the DSB using the supplied DNA fragment
as a template. The HR machinery searches for homology between the damaged chromosome and the extrachromosomal fragment and copies the sequence of the
fragment between the two broken ends of the chromosome, regardless of whether the fragment contains the
original sequence. If the subject is homozygous for the
target allele, the eciency of the technique is reduced
since the undamaged copy of the allele may be used as a
template for repair instead of the supplied fragment.

3.3 Gene therapy

The success of gene therapy depends on the ecient insertion of therapeutic genes at the appropriate
chromosomal target sites within the human genome,
without causing cell injury, oncogenic mutations or an
immune response. The construction of plasmid vectors
is simple and straightforward. Custom-designed ZFNs
that combine the non-specic cleavage domain (N) of
FokI endonuclease with zinc-nger proteins (ZFPs) offer a general way to deliver a site-specic DSB to the
genome, and stimulate local homologous recombination
by several orders of magnitude. This makes targeted gene
correction or genome editing a viable option in human
cells. Since ZFN-encoding plasmids could be used to
transiently express ZFNs to target a DSB to a specic
gene locus in human cells, they oer an excellent way
for targeted delivery of the therapeutic genes to a preselected chromosomal site. The ZFN-encoding plasmid-

3
based approach has the potential to circumvent all the
problems associated with the viral delivery of therapeutic genes.[35] The rst therapeutic applications of ZFNs
are likely to involve ex vivo therapy using a patients own
stem cells. After editing the stem cell genome, the cells
could be expanded in culture and reinserted into the patient to produce dierentiated cells with corrected functions. The initial targets will likely include the causes of
monogenic diseases such as the IL2R gene and the bglobin gene for gene correction and CCR5 gene for mutagenesis and disablement.[32]

4
4.1

Potential Problems
O-target Cleavage

If the zinc nger domains are not specic enough for their
target site or they do not target a unique site within the
genome of interest, o-target cleavage may occur. Such
o-target cleavage may lead to the production of enough
double-strand breaks to overwhelm the repair machinery and, as a consequence, yield chromosomal rearrangements and/or cell death. O-target cleavage events may
also promote random integration of donor DNA.[32] Two
separate methods have been demonstrated to decrease
o-target cleavage for 3-nger ZFNs that target two adjacent 9-basepair sites.[36] Other groups use ZFNs with
4, 5 or 6 zinc ngers that target longer and presumably
rarer sites and such ZFNs could theoretically yield less
o-target activity. A comparison of a pair of 3-nger
ZFNs and a pair of 4-nger ZFNs detected o-target
cleavage in human cells at 31 loci for the 3-nger ZFNs
and at 9 loci for the 4-nger ZFNs.[37] Whole genome sequencing of C. elegans modied with a pair of 5-nger
ZFNs found only the intended modication and a deletion at a site unrelated to the ZFN site indicating this
pair of ZFNs was capable of targeting a unique site in the
C. elegans genome.[20]

4.2

Immunogenicity

For more details on this topic, see Adaptive immune

response.
As with many foreign proteins inserted into the human
body, there is a risk of an immunological response against
the therapeutic agent and the cells in which it is active.
Since the protein will need to be expressed only transiently, however, the time over which a response may develop is short.[32] Liu et al respectively target ZFNickases to the endogenous b-casein(CSN2) locus stimulates lysostaphin and human lysozyme gene addition by
homology-directed repair and derive secrete lysostaphin
cows.[38] [39]

5 Prospects
The ability to precisely manipulate the genomes of plants,
animals and insects has numerous applications in basic research, agriculture, and human therapeutics. Using ZFNs
to modify endogenous genes has traditionally been a difcult task due mainly to the challenge of generating zinc
nger domains that target the desired sequence with sufcient specicity. Improved methods of engineering zinc
nger domains and the availability of ZFNs from a commercial supplier now put this technology in the hands of
increasing numbers of researchers. Several groups are
also developing other types of engineered nucleases including engineered homing endonucleases [40] [41] and nucleases based on engineered TAL eectors. [42] [43] TAL
eector nucleases (TALENs) are particularly interesting
because TAL eectors appear to be very simple to engineer [44] [45] and TALENs can be used to target endogenous loci in human cells.[46] But to date no one has reported the isolation of clonal cell lines or transgenic organisms using such reagents. One type of ZFN, known
as SB-728-T, has been tested for potential application in
the treatment of HIV.[47]

6 Zinc-nger Nickases
Zinc-nger nickases (ZFNickases) are created by inactivating the catalytic activity of one ZFN monomer in
the ZFN dimer required for double-strand cleavage.[48]
ZFNickases demonstrate strand-specic nicking activity
in vitro and thus provide for highly specic single-strand
breaks in DNA.[48] These SSBs undergo the same cellular
mechanisms for DNA that ZFNs exploit, but they show a
signicantly reduced frequency of mutagenic NHEJ repairs at their target nicking site. This reduction provides
a bias for HR-mediated gene modications. ZFNickases
can induce targeted HR in cultured human and livestock
cells, although at lower levels than corresponding ZFNs
from which they were derived because nicks can be repaired without genetic alteration.[49] [38] A major limitation of ZFN-mediated gene modications is the competition between NHEJ and HR repair pathways. Regardless
of the presence of a DNA donor construct, both repair
mechanisms can be activated following DSBs induced by
ZFNs. Thus, ZFNickases is the rst plausible attempt at
engineering a method to favor the HR method of DNA
repair as opposed to the error-prone NHEJ repair. By reducing NHEJ repairs, ZFNickases can thereby reduce the
spectrum of unwanted o-target alterations. The ease by
which ZFNickases can be derive from ZFNs provides a
great platform for further studies regarding the optimization of ZFNickases and possibly increasing their levels
of targeted HR while still maintain their reduced NHEJ
frequency.

7 ZINC FINGER NUCLEASE TREATMENT OF HIV

Zinc Finger Nuclease Treatment

of HIV

A. Targeting and Editing Host Cellular Co-Receptors for

HIV

specicity (due to their short recognition sequences) unfortunately, rendered them toxic to the host genome. The
latter-potential host-genome toxicity posed by the raw
bacteria-derived REases limited their application to exvivo modalities for HIV prevention, namely synthetic or
live microbicides. Subsequently, however, the unique
specicity oered by ZFNs was quickly recognized and
harnessed, paving way for a novel strategy for attacking HIV in-vivo (through target mutagenesis of proviral
HIV DNA) that is similar to the manner by which bacteria equipped with R-M systems do, to disable the foreign DNAs of in-coming phage-genomes. Because latent proviral HIV DNA resident in resting memory CD4
cells forms the major barrier to the eradication of HIV
by highly active antiviral therapy (HAART), it is speculated that this approach may oer a 'functional cure for
HIV. Both ex-vivo (manipulation of stem or autologous
T cell precursors) and in-vivo delivery platforms are being explored. It is also hoped that, when applied to nonHIV infected persons, this strategy could oer a genomic
vaccine against HIV and other viruses. Similar work is
ongoing for high-risk HPVs (with the intent of reversing cervical neoplasia) [60] as well as with HSV-2 (with
the goal of achieving a complete cure for genital herpes)

Since antiretroviral therapy requires a lifelong treatment

regimen, research to nd more permanent cures for HIV
infection is currently underway.[50] It is possible to synthesize zinc nger nucleotides with zinc nger components that selectively (almost selectively) bind to specic
portions of DNA. It has also been observed that 20% of
the Caucasian population possess what is referred to as
the CCR5-32 mutation (frequency of 0.0808 for homozygous allele) that prevents the CCR5 protein, which
is the main means of viral access into the cell, from being
expressed on the surface of their T-cells.[51][52][53][54][55]
Individuals who are homozygous for this mutation are immune to HIV strains that utilize the CCR5 receptor in order to gain access to the cell while those who are heterozygous for this mutation have been found to have reduced
plasma viral load in addition to a delayed progression to
AIDS.[56] By combining these facts, researchers have proposed a novel method of treatment for HIV. This method [61][62][63][64][65][66][67][68][69][70][71]
attempts to treat the infection by introducing the CCR532 mutation and, as a consequence, resulting in the expression of nonfunctional CCR5 co-receptors on CD4+ T
7.1 Zinc Finger Binding
cells, providing immunity against infection.[1][56][57]
The zinc nger nucleases that have been synthesized
for this treatment are manufactured by combining FokI
Type II restriction endonucleases with engineered zinc
ngers.[1][58] The number of zinc ngers attached to the
endonuclease controls the specicity of the ZFN since
they are engineered to preferentially bind to specic base
sequences in DNA. Each ZFN is made up of multiple zinc
ngers and one nuclease enzyme.[1]

The exact constitution of the ZFNs that are to be used

to treat HIV is still unknown. The binding of ZFNs for
the alteration of the Zif268 genelink, however, has been
well-studied and is outlined below in order to illustrate
the mechanism by which the zinc nger domain of ZFNs
bind to DNA.[72][73]

The amino terminus of the alpha helix portion of zinc

ngers targets the major grooves of the DNA helix and
B. Targeting Proviral HIV DNA
binds near the CCR5 gene positioning FokI in a suitable
[1][72][73]
A recent and unique application of ZFN-technology to location for DNA cleavage.
treat HIV has emerged whose focus is to target not Zinc ngers are repeated structural protein motifs with
the host genome, but rather proviral HIV DNA, for DNA recognition function that t in the major grooves
mutagenesis.[59] The authors of this work have drawn of DNA.[72] Three zinc ngers are positioned in a semitheir inspiration from the innate defense mechanism circular or C-shaped arrangement.[73] Each zinc nger
against bacteria-infecting-viruses called bacteriophages, is made up of anti-parallel beta sheets and an alpha
present amongst those bacteria endowed with restric- helix, held together by a zinc ion and hydrophobic
tion modication (R-M) systems. These bacteria se- residues.[72][73]
cret a restriction enzyme (REase) that recognizes and
repetitively cleaves around palindromic sequences within The zinc atom is constrained in a tetrahedral conformathe xenogenic DNAs of the bacteriophages or simply tion through the coordination of Cys3, Cys6, His19, and
phages, until the same is disabled. Further support for His23 and Zinc Sulfur bond distance of 2.30 +/- 0.05
Nitrogen bond distances of 2.0 +/this approach resides in the fact that, the human genome Angstroms and Zinc
[73][74][75]
0.05
Angstroms.
comprises in large part remnants of retroviral genomes
that have been inactivated by several mechanisms, some Each zinc nger has an arginine (arg) amino acid proof whose action resembles that of ZFN. It should not truding from the alpha helix, which forms a hydrogen
be surprising, therefore, that the initial work leading to bond with Nitrogen 7 and Oxygen 6 of the guanine (gua)
the application of ZFN technology in this manner re- that is located at the 3 end of the binding site.[72][73][75]
volved around and involved the isolation and testing of The arg-gua bond is stabilized by Aspartic acid from a
HIV/SIV targeting bacteria-derived REases, whose non- 2nd residue, which positions the long chain of arginine

7.4

Limitations

through a hydrogen bond salt bridge interaction.[72][76]

desired nucleotide sequence allows for gene insertion or

[57]
In residue 3 of the 2nd (i.e., middle) zinc nger, modication.
histidine49 forms a hydrogen bond with a co-planar The main DSB repair pathway in mammals (that occurs
guanine in base pair 6. The stacking of Histidine against in the absence of a homologous nucleotide base sequence
Thymine in base pair 5 limits the conformational abil- that can be used by a homologous recombination mechaity of Histidine49 leading to increased specicity for the nism is through non-homologous end joining (NHEJ).[77]
NHEJ, although capable of restoring a damaged gene, is
histidine-guanine hydrogen bond.[72][73]
[77]
DSB are, therefore, introduced into the
At the 6th residue, ngers 1 and 3 have arginine donating error-prone.
gene
until
an
error
in its repair occurs at which point
a pair of charged hydrogen bonds to Nitrogen 7 and OxyZFNs
are
no
longer
able to bind and dimerize and the
gen 6 of guanine at the 5 end enhancing the site recogni[77]
mutation
is
complete.
In order to accelerate this pro[72][73]
tion sequence of zinc ngers.
cess, exonucleases can be introduced to digest the ends of
Contacts with DNA backbone
the strands generated at DSBs.[77]
The Histidine coordinated to the zinc atom, which is also
the seventh residue in the alpha helix of the zinc ngers,
coordinates the Zinc ion through its N and hydrogen
bonds with phosphodiester oxygen through N on the pri- 7.4 Limitations
mary DNA strand.[72][73][76]
In addition to histidine, a conserved arginine on the sec- Increasing the number of zinc ngers increases the speciond beta strand of the zinc ngers makes contact with the city by increasing the number of base pairs that the ZFN
phosphodiester oxygen on the DNA strand.[72][73][76]
can bind to.[1] However too many zinc ngers can lead to
[1]
Also Serine 75 on the third nger hydrogen bonds to the o-target binding and thus osite cleavage. This is due
phosphate between base pairs 7 and 8, as the only back- to an increased likelihood of zinc ngers binding to parts
bone contact with the secondary strand of DNA.[72][73][76] of the genome outside of the gene of interest.
Current ZFN treatments focus on the CCR5 gene as no
known side eects result from altering CCR5.[78] There
7.2 Nuclease Dimerization and Cleavage
are strains of HIV that are able to use CXCR4 to enter the
host cell, bypassing CCR5 altogether.[78] The same gene
It has been discovered that FokI has no intrinsic speci- editing technology has been applied to CXCR4 alone and
city in its cleavage of DNA and that the zinc n- in combination with CCR5 [79][80]
ger recognition domain confers selectivity to zinc nger
Several issues exist with this experimental treatment. One
nucleases.[1][58]
issue lies in ensuring that the desired repair mechanism
Specicity is provided by dimerization, which decreases is the one that is used to repair the DSB following gene
the probability of o-site cleavage. Each set of zinc n- addition.[81] Another issue with the disruption of the
gers is specic to a nucleotide sequence on either side CCR5 gene is that CXCR4-specic or dual-tropic strains
of the targeted gene 5-7 bp separation between nuclease are still able to access the cell.[81] This method can precomponents.[1]
vent the progression of HIV infection.
The dimerization of two ZFNs is required to produce the
necessary double-strand break within the CCR5 gene because the interaction between the FokI enzyme and DNA
is weak.[57] This break is repaired by the natural repair
mechanisms of the cell, specically non-homologous end
joining.[57]

7.3

Introducing the CCR5 Mutation

Introducing genome alterations depends upon either of

the two natural repair mechanisms of a cell: nonhomologous end joining (NHEJ) and homology-directed
repair (HDR).[57] Repair through NHEJ comes about by
the ligation of the end of the broken strands and, upon
the occurrence of an error, can produce small insertions
and deletions. HDR, on the other hand, makes use of a
homologous DNA strand in order to repair and gene and
making use of this repair mechanism and providing the

To employ the ZFNs in clinical settings the following criteria need to be met: i) High specicity of DNA-binding
Correlates with better performance and less toxicity
of ZFNs. Engineered ZFNs take into account positional
and context-dependent eects of zinc ngers to increase
specicity.[82] ii) Enable allosteric activation of FokI once
bound to DNA in order for it to produce only the required
DSB.[82] iii) In order to deliver two dierent zinc nger
nuclease subunits and donor DNA to the cell, the vectors
that are used need to be improved to decrease the risk
of mutagenesis.[82] These include adeno-associated virus
vectors, integrase-decient lentiviral vectors and adenovirus type 5 vectors.[82] iv) Transient expression of ZFNs
would be preferred over permanent expression of these
proteins in order to avoid o-target eects.[82] v) During
gene targeting, genotoxicity associated with high expression of ZFNs might lead to cell apoptosis and thus needs
to be thoroughly veried in vitro and in vivo transformation assays.[82]

7.5

Administration of Treatment

The cells in which the mutations are induced ex vivo are

ltered out from lymphocytes by apheresis to produce
analogous lentiviral engineered CD4+ T-cells.[83] These
are re-infused into the body as a single dose of 1 X 1010
gene modied analogous CD4+ T-cells.[83] A viral vector
is used to deliver the ZFNs that will induce the desired
mutation into the cells. Conditions that promote this process are carefully monitored ensuring the production of
CCR5 strain HIV-resistant T cells.[84]
The Berlin Patient
Timothy Ray Brown, who underwent a bone marrow transplant in 2007 to treat leukemia, had HIV
simultaneously.[85] Soon after the operation the HIV
dropped to undetectable levels.[85] This is a result of the
bone marrow donor being homozygous for the CCR532 mutation.[85] This new mutation conferred a resistance to HIV in the recipient, eventually leading to an
almost complete disappearance of HIV particles in his
body.[85] After nearly 2 years without antiretroviral drug
therapy, HIV could still not be detected in any of his
tissues.[85][86] Though this method has been eective at
reducing the level of infection, the risks associated with
bone marrow transplants outweighs its potential value as
a treatment for HIV.[52]

See also
Genome editing with engineered nucleases
Zinc nger
Gene targeting
Zinc nger protein
Zinc nger chimera
Protein engineering
Genome engineering

References

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10

Further reading

Mandell JG, Barbas CF (July 2006). Zinc Finger Tools: custom DNA-binding domains for
Nucleic
transcription factors and nucleases.
Acids Res. 34 (Web Server issue): W51623.
doi:10.1093/nar/gkl209. PMC 1538883. PMID
16845061.
Porteus MH, Carroll D (August 2005). Gene targeting using zinc nger nucleases. Nat. Biotechnol. 23 (8): 96773. doi:10.1038/nbt1125. PMID
16082368.
Doyon Y, McCammon JM, Miller JC et al.
(June 2008). Heritable Targeted Gene Disruption in Zebrash Using Designed Zinc Finger Nucleases. Nat. Biotechnol. 26 (6): 7028.
doi:10.1038/nbt1409. PMC 2674762. PMID
18500334.

EXTERNAL LINKS

Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe

SA (June 2008). Targeted gene inactivation in zebrash using engineered zinc nger nucleases. Nat.
Biotechnol. 26 (6): 695701. doi:10.1038/nbt1398.
PMC 2502069. PMID 18500337.

11 External links
Zinc nger selector
Zinc Finger Consortium website
Zinc Finger Consortium materials from Addgene
A commercial supplier of ZFNs

11

12
12.1

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File:Repair_outcomes_of_a_genomic_double-strand_break_for_ZFN_cleavage.jpg
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wikipedia/commons/d/de/Repair_outcomes_of_a_genomic_double-strand_break_for_ZFN_cleavage.jpg License: CC BY-SA 3.0
Contributors: Carroll, D. Genome engineering with zinc-nger nucleases. Genetics Society of America, 2011, 188(4), pp 773-782.
Original artist: Dana Carroll

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