REVIEW

Zinc-finger nucleases: a powerful tool for geneticengineering of animals

Severine Remy Æ Laurent Tesson Æ Severine Menoret Æ

Claire Usal Æ Andrew M. Scharenberg Æ Ignacio Anegon

Received: 13 August 2009 / Accepted: 10 September 2009 / Published online: 26 September 2009

� Springer Science+Business Media B.V. 2009

Abstract The generation of genetically modified

animals or plants with gene-targeted deletions or

modifications is a powerful tool to analyze gene

function, study disease and produce organisms of

economical interest. Until recently, the generation of

animals with gene targeted manipulations has been

accomplished by homologous recombination (HR) in

embryonic stem (ES) cells or cloning through nuclear

transfer and has been limited to a few species.

Recently, a new technology based on the use of gene-

targeted zinc-finger nucleases (ZFNs) was developed

and used for the generation of organisms with gene-

targeted deletions and/or modifications when com-

bined with HR. ZFNs have been used to generate

modified organisms such as plants, Drosophila, zebra

fish and rats with gene-targeted mutations. This

perspective manuscript is a short review on the use

of ZFNs for the genetic engineering of plants and

animals, with particular emphasis on our recent work

involving rats. We also discuss the application of

other targeted nucleases, including homing endonu-

cleases. Microinjection of plasmid or mRNA for

ZFNs into rat embryos allowed targeted, rapid,

complete, permanent and heritable disruption of

endogenous loci. The application of ZFNs to generate

gene-targeted knockouts in species where ES cells or

cloning techniques are not available is an important

new development to answer fundamental biological

questions and develop models of economical interest

such as for the production of humanized antibodies.

Further refinements of ZFN technology in combina-

tion with HR may allow knock-ins in early embryos

even in species where ES cells or cloning techniques

are available.

Keywords Zinc finger nucleases �

Targeted transgenesis � Rat � Gene knockout �

Homologous recombination � Animal models

Introduction

Techniques available for genome modifications are

either random or targeted. Gene targeting involves HR

using ES cells or nuclear transfer and thus is limited to

S. Remy � L. Tesson � S. Menoret � C. Usal �

I. Anegon (&)

INSERM, U643, 44093 Nantes, France

e-mail: ianegon@nantes.inserm.fr

S. Remy � L. Tesson � S. Menoret � C. Usal � I. Anegon

CHU Nantes, Institut de Transplantation et de Recherche

en Transplantation, ITERT, 44093 Nantes, France

S. Remy � L. Tesson � S. Menoret � C. Usal � I. Anegon

Universite de Nantes, Faculte de Medecine,

44093 Nantes, France

A. M. Scharenberg

Seattle Children’s Research Institute, 1900 9th Avenue,

Seattle, WA 98103, USA

123

Transgenic Res (2010) 19:363–371

DOI 10.1007/s11248-009-9323-7

species in which appropriate cells are available, i.e.,

mice, pigs and cows. Random mutagenesis involves

the use of chemicals or transposons. Although these

techniques have been used for the generation of many

useful mutants in many different species, they are

cumbersome and expensive and do not allow targeted

modification of specific genes. Therefore, the gener-

ation of transgenic organisms with gene-targeted

modifications has been hampered in most species by

the lack of appropriate technologies.

The rat is an important biomedical research model

whose utility has been significantly hampered by a

lack of technologies for targeted genome modification

(Aitman et al. 2008; Jacob 2009). Transgenic rats have

been generated using microinjection of DNA (Char-

reau et al. 1996; Menoret et al. 2009; Mullins et al.

1990) or lentiviral vectors (Lois et al. 2002; Pfeifer

2006; Remy et al. 2009). Over 195 transgenic or

mutant rats have been produced using chemicals (Zan

et al. 2003) or transposons (Kitada et al. 2007; Tesson

et al. 2005) (for a complete list see: http://www.ifr26.

univ-nantes.fr/ITERT/transgenese-rat/liste_rats.php).

Rat cloning using nuclear transfer (Cozzi et al. 2009;

Zhou et al. 2003) as well as the generation of rat ES

cells (Buehr et al. 2008; Li et al. 2008), induced plu-

ripotent stem cells (iPS) (Kawamata and Ochiya 2009;

Ueda et al. 2008) and spermatogonial stem cells (SSC)

(Shinohara et al. 2006) have been described recently,

but to date the cells used for these procedures have not

been demonstrated to be amenable to any form of

targeted gene modification.

This perspective is a follow-up to our recent

publication, describing the first generation of knockout

rats using a newly developed technique that applies

zinc-finger nucleases (ZFNs) in rat embryos (Geurts

et al. 2009a, b). We discuss our approach and the

present state of the field, and highlight important issues

for investigators to consider in applying nuclease-

mediated gene targeting to animal models.

Zinc finger nucleases and related technologies

for targeted genetic modification

ZFNs are hybrid molecules composed of a designed

polymeric zinc finger domain specific for a DNA

target sequence and a FokI nuclease cleavage domain

(Kandavelou and Chandrasegaran 2009; Kim et al.

1997; Miller et al. 2007; Porteus 2008; Santiago et al.

2008; Shukla et al. 2009; Szczepek et al. 2007). FokI

requires dimerization to cut DNA. The binding of two

heterodimers of designed ZFN-FokI hybrid mole-

cules to two contiguous target sequences in each

DNA strand separated by a 6 base-pair cleavage site

results in FokI dimerization and subsequent DNA

cleavage.

The specificity of ZFN’s is determined by their

polymeric zinc finger domains, the DNA binding

properties of which are generated through modular

assembly of individual zinc fingers [reviewed in

(Beerli and Barbas 2002; Pabo et al. 2001)]. Two

major platforms exist for generating polymeric zinc

fingers with defined specificities: a proprietary plat-

form developed by Sangamo Biosciences (Isalan et al.

2001; Urnov et al. 2005), and the OPEN platform

developed by the Zinc Finger Consortium (Maeder

et al. 2008; Sander et al. 2007;Wright et al. 2006). Both

are now accessible for transgenic animal research

purposes: Sangamo has partnered with Sigma to sell

pre-assembled ZFN’s via the Compozr program

(http://www.compozrzfn.com/), while the OPEN plat-

form (http://www.addgene.org/zfc; www.zincfingers.

org/software-tools.htm) makes their modular assembly

zinc finger pools and reagents freely available.

Induction of a DNA double strand break by a ZFN

results in the activation of a cellular response known

as the DNA damage response. A double strand break

can be repaired in two different ways (Fig. 1). Non-

homologous end joining (NHEJ) generates short

insertions or deletions at the cleavage site. Repair

by HR using a DNA template results in gene knock-

ins that are either a perfect repair or, if a modified

template is introduced, sequence replacement.

Zinc-finger nucleases (ZFNs) have been used to

generate gene-targeted knockin in cultured cells

(Bibikova et al. 2003), including human ES and iPS

cells (Hockemeyer et al. 2009), plants (Cai et al. 2009;

Townsend et al. 2009) and Drosophila (Beumer et al.

2008; Bibikova et al. 2002; Bozas et al. 2009) as well

as gene-targeted knockouts in Xenopus, C. elegans

(Carroll et al. 2008), and zebra fish (Doyon et al. 2008;

Foley et al. 2009). ZF fused to transcription activators

have also been used to target specific promoter

sequences and induce gene expression in transgenic

mice (Mattei et al. 2007). ZFNs have also been used to

induce gene-targeted knock-ins in cells in the context

of genome editing for gene therapy (Lombardo et al.

2007; Moehle et al. 2007; Perez et al. 2008). Related

364 Transgenic Res (2010) 19:363–371

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technologies such as homing endonucleases utilize a

different DNA cleavage mechanism, but the resulting

DNA double strand break is resolved through the same

mechanisms and engineered homing endonucleases

(Monnat et al. 2008; Paques and Duchateau 2007;

Stoddard et al. 2008) or synthetic nucleases (Cannata

et al. 2008) can be applied in the same manner as

ZFNs. The higher engineering barriers to generating

novel homing endonucleases has so far limited their

application to a few plant (Yang et al. 2009) and

human applications (Grizot et al. 2009; Paques and

Duchateau 2007), although recent progress should

allow more widespread application as the engineering

barriers to developing novel homing endonucleases

are reduced (Arnould et al. 2006; Ashworth et al.

2006; Takeuchi et al. 2009; Volna et al. 2007).

Zinc finger nucleases-mediated targeted gene

modification in rats

We have recently described that expression of ZFNs

in rat embryos resulted in a high frequency of

mutated live offspring with complete, permanent and

heritable disruptions of the immunoglobulin (Ig)

heavy chain l locus (IgM) (Geurts et al. 2009a, b).

Since IgM cell membrane expression on B lympho-

cytes is needed for their survival and the expression

of all the other Ig isotypes (Kitamura et al. 1991)

these IgM-knockout rats should be mature B lym-

phocytes- and heavy chain Ig-deficient. These Ig-

deficient rats would be useful models for the analysis

of the role of antibodies in a variety of pathophys-

iological situations. Additionally, these Ig-deficient

rats could be crossed with transgenic rats for the

human heavy and light chain Ig and thus constitute a

platform for the production of human antibodies

directed against any target with potential clinical

interest. In the same manuscript (Geurts et al. 2009b),

the group of H. J. Jacobs generated knockout rats for

a GFP transgene inserted into the genome by

lentiviral transgenesis and an endogenous gene,

Rab38. Mutants were isolated with three different

rat inbred or outbred strains.

For our approach to generating gene-targeted rats,

ZFNs targeted to the IgM locus (IgM ZFNs) were

cloned in cis into an expression plasmid as two open

reading frames linked by the self-cleaving peptide

T2A under the transcriptional control of the CAG

promoter and also containing 30 polyA sequences

(Fig. 2) (Geurts et al. 2009a, b). The ZFN-encoding

circular plasmid DNA or mRNA were delivered by

Target sequence

(exon of coding gene)

Non Homologous End-Joining

(NHEJ)

gene disruption in ~70 % of NHEJ

events by out of frame deletions

Homologous Repair

(Template)

Targeted

insertion

Double strand break

ZFNs

ZFNs

Fig. 1 ZFN mediated targeted genome modification relies on

cellular DNA repair pathways. ZFNs recognize specific gene

sequence in each DNA strand of a given loci and are separated

by a 6 base pair sequence where the FokI nuclease will

generate a double strand break. The cellular DNA repair the

break by non-homologous end joining, which is imperfect and

generates in *70% of the cases gene deletions due to out of

frame deletions of variable lengths, or by homologous

recombination when a repair sequence is introduced into the

cell together with the ZFNs

Transgenic Res (2010) 19:363–371 365

123

pronuclear or intracytoplasmic injection (Fig. 2) in

order to limit the duration of ZFN expression. ZFNs

were injected at different concentrations and the

percentage of mutated embryos (5–75%) obtained

was directly correlated to the concentration of

injected ZFN vector [Table 1; (Geurts et al. 2009a,

b)]. Sequence analysis of 22 founders revealed

deletions ranging from 3 to 224 base pairs. Some of

the animals showed more than one deletion, indicat-

ing ZFNs acted not only at the zygote stage but also

at later time points of embryonic development. While

most of the founders showed one normal IgM allele,

one animal carried biallelic mutations in IgM. ZFN-

mediated gene disruption also demonstrated high

fidelity for each target sequence as no ZFN-induced

mutations were detected in target-gene-disrupted

animals at any of 12 predicted ZFN off-target sites.

Importantly, after breeding to wild-type animals, 1/1

GFP and 5 out of 7 IgM mutant rats transmitted

mutated alleles through the germline (Table 2).

Phenotypic analysis of animals from one of these

IgM-knockout rat lines showed undetectable or very

low serum levels of IgM, IgG, IgA and IgE (man-

uscript in preparation), confirming that the deletion in

the IgM CH1 region resulted in Ig deficiency and not

in alternative splicing and generation of Ig of shorter

l heavy chain length, as previously reported in mice

(Zou et al. 2001).

Taken together, our application of plasmid or

mRNA ZFN-mediated gene inactivation in rats

resulted in a single-step whole rat gene knockout

that was targeted, rapid, complete and permanent.

ZFNs were expressed transiently and showed mini-

mal off-target effects in both inbred and outbred rat

strains. These Ig-deficient rats should be a useful

biomedical research model.

Further work is ongoing to knockout the rat Ig J,

kappa and lambda chain loci, with the plan to cross the

resulting strains with each other and with transgenic

rats generated with BACs containing the sequences for

human Ig heavy or light chains, thus generating rats

with fully humanized humoral immune responses.

The relative high efficiency of our procedure

supports the possibility of generating whole animal

knockin rats by HR resulting from the simultaneous

delivering into the zygote of both a nuclease and the

donor-additive DNA sequence. In Drosophila, HR

using ZFNs was more efficient when using the circular

form of the donor DNA as compared to the linear one

(Beumer et al. 2008); linear DNA may be more

degraded by nucleases or concatenated and rendered

less capable of HR. Circular versus linear forms of the

donor DNA will have to be tested in mammalian

embryos.Also inDrosophila, deficiency ofDNA ligase

IV, part of the NHEJ mechanism, increased HR

dramatically (Beumer et al. 2008) but DNA ligase IV

deficiency is lethal in mice (Barnes et al. 1998).

Mutation of genes of other proteins involved in NHEJ

and compatible with embryo survival may be a major

future objective to facilitate the use of ZFNs or other

genes to direct HR in mammal zygotes.

Along this line, nucleases could be used to direct

HR to target integration of a new transgene into a

•

•

CAGp ZFN 1-2A-ZFN 2 pA

Rat one-cell embryo

Tail biopsy and DNA analysis for ZFN activity and

sequence

ZFN1-ZFN2

DNA expression vector mRNA (capped and polyA)

Transfer to pseudopregnant

females

If only one allele targeted cross-breed descendence to obtain

homozygous deficient animals

5 months

Fig. 2 Procedure for the generation of knockout rats. The pair

of ZFNs for IgM were assembled in cis in an expression

plasmid separated by a 2A self-splicing sequence (allowing

separate translation from one transcript), the CAG promoter

(CAGp, which is expressed in one cell embryos) and a polyA

sequence (2.87 Kb total). The ZFNs were microinjected either

as circular plasmid (to avoid genomic insertion) or as mRNA

following in vitro transcription. The plasmid DNA was

microinjected into the male pronuclei and the mRNA either

in the male pronuclei or in the cytoplasm. Surviving embryos

were transferred to foster mothers, the DNA from newborns

tail biopsies were analyzed using the Cel-I assay to detect

mutations and confirmed by DNA sequencing. DNA sequenc-

ing will reveal whether the animal is mutated in one or both

alleles and whether the mutation is unique or multiple.

Heterozygous mutated animals are breed with wild-type

animals, the offspring is analyzed to confirm the transmission

of the mutation (at this stage only one mutation) and to cross-

breed to obtain an homozygous knockout. The whole process

can be done in 5 months

366 Transgenic Res (2010) 19:363–371

123

permissive locus that possesses ubiquitous or tissue-

restricted expression, as has been accomplished via

HR in ES cells (Bronson et al. 1996). In this way, any

transgene integrated by HR in such a locus would

reproduce its expression and site-dependent epige-

netic modulation of transgene expression would be

avoided.

Other technical possibilities include the use of

pairs of nucleases directed to two different sequences

of a gene separated by large stretches of DNA in

order to generate long deletions.

Potential barriers for the application

of nuclease-induced genetic modifications

While application of nuclease-mediated gene target-

ing in organisms ranging from plants, to insects, to

Xenopus to zebrafish and now mammals has now

been shown to be possible, a major open question is

how widely nuclease technologies can be success-

fully applied in more complex organisms.

Zinc-finger nucleases (ZFNs), while intended to

make a double strand break at a specific target site,

are known to be promiscuous due to the nature of the

FokI endonuclease cleavage domain. The FokI

endonuclease cleavage domain must dimerize to

create an active endonuclease complex able to

generate a double strand break, and in its native

form is capable of doing so as homodimers even

when not bound to DNA (Mani et al. 2005), albeit

weakly. Even when newer generation of heterodimer-

forced FokI domains are utilized (Geurts et al. 2009b;

Szczepek et al. 2007), expression of these nucleases

in a cell may result in not only the intended, but also

other double strand breaks and consequent mutations.

In this regard, the manifestation of toxicities related

to ZFN injection into zebrafish embryos (Foley et al.

2009) provides confirmatory evidence that ZFN can

induce double strand breaks even at non-canonical

sites. This is the reason why present strategies,

including our application of ZFN’s in rats, favor

short-term expression of ZFNs rather than longer

term expression that would provide additional time

for off target cleavage to lead to additional mutations

and toxic effects. Investigators interested in utilizing

ZFN-mediated gene targeting must thus consider how

best to manage mutations generated in sequences

other than the ones targeted: either, specific tech-

niques may be applied to detect them (Geurts et al.

2009b; Perez et al. 2008), or crossbreeding may be

pursued to eliminate them. To what degree off target

cleavage and consequent mis-targeting of mutations

will apply to engineered homing endonucleases is not

presently known.

DNA double strand breaks are recognized by a

complex machinery that creates epigenetic modifica-

tions extending for many kilobases in either direction

from the DNA double strand break, the recruitment

and assembly of an array of DNA repair proteins into

Table 1 Transgenic

efficiency of microinjection

of ZFNs for rat IgM

Plasmid circular plasmid to

avoid genomic insertion,

mRNA in vitro translated,

PNI pronuclear injection

Construct Route Dose

(ng/ml)

Transferred embryos

% (total injected)

Newborns %

(total transferred)

Mutants %

(total borns)

Plasmid PNI 10 80.9 (609) 11 (493) 11.1 (54)

Plasmid PNI 2 77.3 (605) 17.5 (468) 9.8 (82)

Plasmid PNI 0.4 82.8(511) 14.7 (423) 6.5 (62)

mRNA PNI 10 55.9 (186) 13.5 (104) 28.6 (14)

mRNA PNI 2 60.5 (832) 19.1(503) 11.5 (96)

mRNA PNI 0.4 64.5 (183) 16.1 (118) 5.3 (19)

mRNA Cytoplasmic 10 72 (272) 2 (197) 75 (4)

mRNA Cytoplasmic 2 68 (197) 12.7 (134) 11.7 (17)

Table 2 IgM mutant germ line transmission

Founder No. of

offspring

No. of mutants

(% of offspring)

0019 7 5 (71)

0046 6 1 (17)

0008 5 0 (0)

0006 6 3 (50)

0119 0 0

4.1 12 1

9.2 40 17

Transgenic Res (2010) 19:363–371 367

123

a repair complex, and signaling to cell cycle regula-

tory mechanisms to slow or halt cell cycle progres-

sion so as to prevent unrepaired damage from being

propagated to future generations of cells (Batista

et al. 2009; Donigan and Sweasy 2009; Pandita and

Richardson 2009). The extent of epigenetic modifi-

cations and cell cycle alterations accompanying

induction of a DNA double strand break and their

influence on cell physiology may also vary according

to the gene expression pattern in a given cell. In the

context of a germ cell/oocyte/embryo a DNA double

strand break could result in substantial chromosomal

instability and thus prevent further developmental

progression (e.g., Lo et al. 2002).

NHEJ is the dominant DNA repair mechanism in

somatic cells and HR tends to be upregulated in

mouse ES cells (Derijck et al. 2008). Whether NHEJ-

or HR-mediated resolution of a nuclease-induced

break is favored in early embryos and how NHEJ- or

HR can be manipulated are important areas for future

investigation.

Differences in response to DNA damage between

organisms may also hinder application of nuclease-

mediated gene targeting at the embryo or other stages

of development. Data from induction of double strand

breaks in gonocytes and spermatogonial precursors

via gamma irradiation suggests that such cells are

highly sensitive to induction of apoptosis via double

strand breaks (e.g., Forand et al. 2009), representing a

mechanism through which an organism prevents

genetic changes resulting from double strand breaks

from being transmitted to its offspring. An additional

instructive example is that zebrafish have been shown

to have a higher overall DNA repair capacity than

mice or humans (Sussman 2007). Thus, the effect of a

double strand break during zebrafish embryonic

development would be expected to be less disruptive

versus mouse or rat embryonic development, which

along with the reduced complexity of zebrafish

embryonic development, renders results with nucle-

ase-mediated targeting difficult to extrapolate from

one species to another.

For application to a given organism, the complex-

ity of the embryonic developmental process is also

likely to be an important influence on success. Like

other vertebrates, the developmental program through

which a mammalian embryo develops into a viable

fetus is dependent on complex genetic and epigenetic

processes involving hundreds, if not thousands, of

genes and non-coding RNAs. However, a mammalian

embryo must not only manage its own development,

it must do so in conjunction with a placenta which is

in turn interacting with the maternal body and

immune system. Mammalian embryonic develop-

ment is thus significantly more complex than other

vertebrates, as it depends on precise quantitative

regulation, timing and coordinated expression of each

involved transcriptional unit for both appropriate

somatic development as well as the coordination of

interactions between the placenta and the maternal

environment (Phillips and McKinnon 2007). This

complexity represents another significant barrier to

extrapolation of results from insects, Xenopus, or

zebrafish to mammalian embryos, as the DNA

damage response induced by even a single DNA

double strand break would have multiple additional

opportunities to disrupt development.

A final issue for investigators contemplating the

use of nuclease-mediated gene targeting is that

success at any single locus may not predict success

at other loci—variability in gene targeting success at

different loci is a well known phenomenon in murine

ES cells. Thus, success at any given loci in any given

organism, does not translate to an obvious potential

for success in gene targeting in the same organism in

another system—i.e., success in targeting the zebra-

fish Ig locus could not be construed as predictive at

success in targeting the Ig locus in the rat, and success

in the rat may likewise not be extrapolated to other

mammals (Danilova et al. 2005). Even our success at

multiple loci in rats does not guarantee success at

other loci. A major issue in this regard is that the

capacity of a nuclease to hit an intended target site

may be subject to the epigenetic status of the locus

containing the target site—e.g., in the most simplistic

case, is a target in euchromatin or heterochromatin?

Thus, even if a given target is accessible to a targeted

nuclease in the context of one cell type, one could not

know that the same target would be accessible with

comparable efficiency, or even at all, to the same

nuclease in another context. This would be a partic-

ular concern for extrapolating data from cultured cell

models to primary cells of any type, as cultured cell

models are often tumor derived, and are notorious for

accumulating genetic and epigenetic changes over

time in culture. In addition, due to an unknown

efficiency of cleavage, the rapid proliferation rate of

early development and cell cycle alterations caused by

368 Transgenic Res (2010) 19:363–371

123

double strand break induction, the likelihood of

cleavage of a specific locus in early development is

unknowable prior to performing the experiment in the

specific embryos/oocytes/germ cell system of interest.

Conclusions

In conclusion, ZFNs, homing endonucleases, and

related nuclease technologies represent powerful and

versatile new tools in the genetic engineering toolkit,

including gene therapy (Carroll 2008). If the objec-

tive is to generate targeted-gene knockouts, ZFNs in

particular but possibly also other nucleases-mediated

targeted gene modification are not only crucial in

species where ES cells or cloning by NT are not

available, but are a real alternative to these other

technologies in terms of speed, cost and labor in

species in which they are available. If the objective is

to generate gene-additions by HR, nuclease-mediated

gene targeting has already been shown in non-

mammal species to be indispensable and has the

potential for widespread application for this purpose

in mammals as an alternative to ES cells or cloning

by NT.

Acknowledgments This work was in part funded by the

Region Pays de la Loire through Biogenouest and IMBIO

programs as well as by the IBiSA program, and NIH grant

UL1DE019582 to AMS.

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