TIBTEC-1063; No.
of Pages 9
Review
Site-directed nucleases: a paradigm
shift in predictable, knowledge-based
plant breeding
Nancy Podevin1*, Howard V. Davies2, Frank Hartung3,
Fabien Nogué4, and Josep M. Casacuberta5
1
The European Food Safety Authority (EFSA), Via Carlo Magno 1A, Parma, Italy
2
The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK
3
Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Biosafety in Plant Biotechnology,
Erwin-Baur-Str. 27, D-06484 Quedlinburg, Germany
4
INRA AgroParisTech, IJPB, UMR 1318, INRA Centre de Versailles, route de Saint Cyr, 78026 Versailles CEDEX, France
5
Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Campus UAB, Cerdanyola del Vallès 08193 Barcelona, Spain
Conventional plant breeding exploits existing genetic
variability and introduces new variability by mutagene-
sis. This has proven highly successful in securing food
supplies for an ever-growing human population. The use
of genetically modified plants is a complementary ap-
proach but all plant breeding techniques have limita-
tions. Here, we discuss how the recent evolution of
targeted mutagenesis and DNA insertion techniques
based on tailor-made site-directed nucleases (SDNs)
provides opportunities to overcome such limitations.
Plant breeding companies are exploiting SDNs to devel-
op a new generation of crops with new and improved
traits. Nevertheless, some technical limitations as well
as significant uncertainties on the regulatory status of
SDNs may challenge their use for commercial plant
breeding.
SDNs in plant breeding
Plants and their derived food products are the cornerstone of
human nutrition, and since Neolithic times plant breeding
has increased yields and improved quality and nutritional
value. Plant breeding has therefore delivered to feed an
ever-growing population, although significant challenges
remain [1]. Plant breeding is based on the introduction of
genetic variability, historically using a ‘cross the best with
the best and hope for the best’ approach but development in
contemporary genetics and genomics has driven resurgence
in the search for novel sources of variation from germplasm
collections (wild species and landraces). Human interven-
tion has also introduced new variation using agents such as
irradiation and chemical mutagenic compounds which, in
the past 20 years, has been complemented by transgenesis,
Corresponding author: Casacuberta, J.M. (josep.casacuberta@cragenomica.es)
*
Current address: European Molecular Biology Laboratory, Meyerhofstrasse 1,
Heidelberg, Germany.
Keywords: site-directed nuclease; zinc-finger nucleases (ZFN); transcription activator-
like effector nucleases (TALEN); meganuclease; plant breeding; genetically modified
organism (GMO).
0167-7799/$ – see front matter
ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2013.
03.004
resulting in the production of genetically modified plants
(GMPs, see Glossary).
Modern agriculture is challenged with feeding a global
population (predicted to reach 9 billion by 2025) but with
reduced availability of natural resources (productive land,
water, and nutrients) and concurrent with negative
impacts of changing climates (e.g., extreme weather and
new/emerging pests and diseases). Primary production
agriculture and plant breeding will require an expansive
toolkit to meet these challenges because there is no ‘one
size fits all’ solution. Conventional plant breeding can be
limited in providing the genes and alleles required to meet
the challenges, whereas conventional mutation breeding
requires time consuming and expensive screens on large
populations. Moreover, these methods are serendipitous,
which limits their use to the production of plants with gene
knockouts. With respect to transgenesis, one of the limita-
tions is the complexity of the regulatory process for GMPs
Glossary
Double-strand break (DSB): a break in double-stranded DNA in which both
strands have been cleaved. It can originate from byproducts of cell metabolism
such as reactive oxygen species, from environmental exposure to irradiation,
chemical agents, or UV light. DSBs also occur as intermediates in various
biological events, such as V(D)J recombination, transposon movement or
meiotic recombination. See Box 2 for an explanation of the repair mechanisms
for DSBs.
Genetically modified plants (GMPs): see ‘Transgenic’
Homologous recombination (HR): error-free repair of a DSB in DNA, in which
the broken DNA molecule is repaired using a homologous sequence. See Box 4
for details on DNA repair mechanisms.
Indels: Indel is the combined artificial word for either an insertion or a deletion
which results from DNA damage by an error-prone DNA-repair mechanism or
an error during DNA replication.
Nonhomologous end joining (NHEJ): a major pathway of DSB repair, in which
the ends are directly ligated without the need for a homologous template.
NHEJ consists of two subpathways: canonical end-joining (C-EJ) and
alternative end-joining (A-EJ). See Box 4 for details on DNA repair mechan-
isms.
Site-directed nucleases (SDNs): nucleases that can be directed to specific DNA
sequences through a DNA binding activity (see Box 1 for different types of
SDNs).
Transgenic: in the context of this paper, transgenic refers to plants containing a
gene or genes that have been inserted using a genetic modification/
engineering technique, and that are referred to as GMPs.
Trends in Biotechnology xx (2012) 1–9 1
TIBTEC-1063; No. of Pages 9
Review
together with detailed, time-consuming and expensive
safety analysis. Thus, the technology is primarily the
domain of big companies developing profitable traits in
major crops. Regulatory oversight for GMPs has been
partly justified by the imprecision of the transgenesis
technique (random insertion in the genome and potential
unintended effects). In recent years techniques have been
developed for genome modification based on the use of
SDNs for targeted mutagenesis of genes or targeted inser-
tion of DNA sequences. The precise manipulation of gen-
omes possible with SDN techniques may overcome some of
the constraints associated with conventional or transgenic-
based breeding techniques, although SDN techniques still
have some limitations. Also, uncertainties remain over the
future application of SDN approaches given impending
decisions on their legal status and required regulatory
oversight [2].
In this review we briefly summarise the current knowl-
edge of SDN techniques and their use in plants, together
with their potential use to introduce new traits in commer-
cial crops. Moreover, we compare SDN techniques with
respect to the methods currently used in plant breeding
and discuss the potential safety issues related to the use of
these techniques and the appropriate safety evaluation
and regulatory oversight of the products obtained by these
techniques.
SDNs: modes of action and uses for genome
modification
SDN techniques are based on the introduction of double-
strand breaks (DSBs) at specific locations within the ge-
nome via tailor-made SDNs. SDNs include meganucleases
(MNs), zinc-finger nucleases (ZFNs) and transcription-ac-
tivator like (TAL) effector nucleases (TALENs) (Box 1).
These SDNs have been developed and tested using both
plants and animal systems and they have been used for the
targeted modification of both plant and animal genomes.
The three different types of nucleases have advantages
and disadvantages [3]. The small size of MNs makes them
easier to deliver and their high specificity results in less
damage to the cell (i.e., lower frequency of DSBs at off-
target sites). However, the fact that their DNA binding and
enzymatic activities cannot be easily separated, as well as
the complex array of protein/DNA-specific interactions
they establish, makes them less flexible and more difficult
to engineer [4]. Conversely, the modular nature of the
DNA-binding motifs of ZFNs, and in particular TALENs,
makes them highly flexible, facilitating the engineering of
DNA-binding modules for virtually any sequence of inter-
est. However, a lower specificity has been observed with
ZFNs compared with MNs and TALENs, leading to signif-
icant off-target DSBs and higher levels of cellular damage
[5–8].
The outcome of the DSBs introduced by SDNs will
depend on the mechanism used for DNA repair (Box 2)
and on the presence of homologous DNA to be used as a
repair template (Figure 1).
A large number of different purposes and possible out-
comes exist for SDNs. To simplify their descriptions and
facilitate a discussion on possible regulatory oversight
of derived products, a classification into three different
2
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Box 1. Different types of SDNs (MNs, ZFNs, and TALENs)
MNs
MNs are naturally occurring endodeoxyribonucleases encoded by
mobile introns of a wide range of organisms that are characterised
by a large recognition site (DNA sequences of typically 20–30 bp)
[53]. The best studied MNs and the most widely used in research
and genome engineering include I-SceI (discovered in the mito-
chondria of baker’s yeast Saccharomyces cerevisiae), I-CreI (from
the chloroplasts of the green alga Chlamydomonas reinhardtii) and
I-DmoI (from the archaebacterium Desulfurococcus mobilis) [54].
The DNA-binding site, which contains the catalytic domain, is
composed of two components located at either side of the DNA
cleavage site [53] (see Figure 1 in main text). The use of MNs has
been limited by the repertoire available. However, tailor-made MNs
have been developed either by modifying the specificity of existing
natural MNs through the introduction of a small number of
variations in the amino acid sequence of the natural recognition
site [55], or by fusing protein domains from different enzymes [56].
ZFNs and TALENs
In contrast to MNs, ZFNs and TALENs are chimeric enzymes that
combine a nuclease (frequently based on the FokI enzyme) and a
DNA-binding domain derived from ZF proteins or TAL effectors. A
single finger-like structure of a ZF DNA-binding domain recognises a
specific 3-bp sequence [25,57]. As each ZF binds a different
sequence, they can be linked together as independent modules,
resulting in a peptide that can be designed to bind a predetermined
DNA site [25,57]. They therefore usually target sequences of 9 bp.
The enzyme FokI functions as a dimer, therefore, two ZFN units are
needed to cleave the DNA and the complete recognition site for
ZFNs consists of 18 bp.
The TAL effectors from Xanthomonas are trans-kingdom tran-
scription factors. They are injected by the bacteria into plant cells
where they activate the expression of specific plant genes that aid
bacterial colonisation and spread [58,59]. The DNA-binding domain
of TAL effectors contains 33–35 amino acid sequence repeats which
include a repeat-variable di-residue (RVD) at residues 12 and 13,
determining their specificity in DNA binding [59]. This simple
relation, whereby each repeat binds a specific nucleotide, has
facilitated the engineering of specific DNA-binding domains by
selecting a combination of repeat segments containing the appro-
priate RVD [60,61]. The number of repeats and the sequence of the
RVD determine the length and sequence of the target sequence that
will be recognised.
Future SDN developments
Recently, several groups have exploited the CRISPR (clustered
regularly interspaced short palindromic repeat) immune system of
prokaryotes [62] for successful targeting of the nuclease Cas9
(CRISPR associated 9) by synthesised guide RNAs to specific loci in
eukaryotic genomes [63–65]. This system works in principle like the
above described SDNs but the binding part consists of two small
RNAs that form a partially double-stranded RNA. This guide RNA
works also as an artificially fused chimera and can be designed and
synthesised to target a specific sequence of a given genome. The
cleaving part of this new SDN is the codon optimised Cas9 protein,
which forms a complex with the chimeric guide RNA and introduces
a DSB in the DNA at the specific target side.
groups has been proposed [9] (Figure 1). The SDN-1 tech-
niques use SDNs to generate site-specific random muta-
tions; in most cases via a single DSB that plants repair
mainly by nonhomologous end joining (NHEJ) [10,11]. The
NHEJ mechanism can result in unfaithful repair, creating
small nucleotide deletions and/or insertions (Indels) (Box
2). The SDN-1 techniques can also be designed to introduce
two DSBs, in which case a deletion of the region between
the two target sites can be obtained. Thus deletions of
regulatory regions, exons/introns, a whole gene or even
parts of chromosomes are possible by introducing two
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Review Trends in Biotechnology xxx xxxx, Vol. xxx, No. x

Box 2. DSB repair mechanisms

Most of the pathways involved in DSB repair are well studied and
components involved have been identified in yeast and mammalian
systems. After induction of a DSB (e.g., due to bleomycin, irradia-
tion, or SDN), the break can be repaired by different pathways,
mostly depending on the extent of resection [progressive single-
stranded (ss)DNA degradation] of the 30 DNA end (Figure I). The
pathways are as follows. (i) C-EJ, which is dependent on KU70/80
and requires no homology. This process seals the broken ends and
assures genome stability. (ii) A-EJ, which is independent of KU70/80.
This process can operate after short or long DNA resection, resulting
in shorter or longer deletions. (iii) HR, which needs a long ssDNA
resection process and the loading of RAD51 (RADiation sensitive
51 protein) on ssDNA to facilitate an active search for homologous
DNA [66]. The DSB is occupied first by the MRN (Mre11–Rad50–
Nbs1) complex and KU70/80 and can be repaired by C-EJ. However,
the release of both complexes can lead directly to A-EJ, which needs
only short ssDNA resection but can also work later on extensively
resected ends. A-EJ is highly error prone, resulting in genomic
instability. Alternatively, to maintain genome stability, the HR
pathway can be activated via BRCA2. HR needs an extensive ssDNA
resection step in which EXO1 (Exonuclease 1 protein) and a RECQ-
helicase (Recombination Q helicase protein) [e.g., BLM (Bloom
syndrome protein) or SGS1 (Slow Growth Suppressor protein)] are
crucial [67].

Key:
DSB
Ku70 BLM-Exo1

Ku80 Lig3

MRN CtlP Extensive

resecon
RAD51 Lig4 Short
Parp1 RPA resecon

DNA-PKcs BRCA2

C-EJ A-EJ HR
NHEJ
TRENDS in Biotechnology

Figure I. Repair of a double-strand break (DSB). Ligation of nonhomologous end-joining (NHEJ) does not require sequence homology. For canonical end-joining (C-EJ),
the DNA ends are recognised by Ku70/Ku80 heterodimer and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Activation of the latter allows the recruitment
of ligase 4 for the ligation step. For alternative end-joining (A-EJ), unprotected DNA ends are degraded by the MRN complex revealing complementary
microhomologies that can anneal. Filling in or nicking of the single strand gap completes the end-joining. These last steps are dependent on PARP1 and ligase 3.
Homologous recombination (HR) uses an intact homologous sequence to repair the DSB and is initiated by resection of single-stranded DNA. The MRN complex, in
association with CtIP, initiates resection, which allows the recruitment of other nucleases, for the formation of single-stranded overhangs that are covered by the RPA
protein. BRCA2 will remove RPA and load the RAD51 protein for the invasion and reparation of the injured strand of the homologous sequence. Abbreviations: MRN
complex, Mre11–Rad50–Nbs1 complex; PARP1, Poly (ADP-ribose) polymerase 1; CtIP, CtBP-interacting protein; RPA, Replication protein A; BRCA2, breast cancer type 2
susceptibility protein; RAD51, RADiation sensitive 51 protein.

DSBs at different sites using either one or two SDNs [12–
14]. The induction of two DSBs can also lead to duplica-
tions and inversions of the sequences located between the
two DSBs. SDN-1 also allows the induction of a transloca-
tion event. In plants only the displacement into a targeted
locus of a transgene already present in the plant has been
achieved to date [15].
The SDN-2 techniques are based on the use of an
externally supplied DNA template (usually called donor
DNA) for the repair that proceeds by homologous recombi-
nation (HR) (Box 2). Donor DNA contains the targeted
desired mutations (a few nucleotide substitutions or short
insertion/deletions) but also requires high sequence iden-
tity to the endogenous gene to allow HR. A successful HR
event substitutes the endogenous sequence by the donor
DNA introducing the desired mutations. Thus, modifica-
tions in gene sequences can be targeted, for example, to
repair undesirable spontaneous mutations (targeted gene
correction) or to introduce targeted mutations and new
phenotypes (gene/allele replacement).
Finally, the SDN-3 techniques aim to introduce long DNA
fragments (e.g., transgenes) at a predefined locus using a
donor DNA (that includes the sequence to be introduced
together with flanking DNAs showing homology to the target
locus) in combination with an SDN. This facilitates the
targeted integration of DNA, which otherwise would inte-
grate randomly in the genome at naturally occurring DSBs.
Uses of SDNs to develop next generation plants for
commercialisation
SDNs have been used in plants both for site-specific muta-
genesis (SDN-1 and SDN-2) and for targeted insertion of
foreign DNA (SDN-3) [16]. Model plants have usually been
used to develop this technique, but SDNs are increasingly
being used in crop plants and an important number of
patents covering these techniques and their uses in plants
already exist [9,17]. Here, we briefly review the recent
advances of the use of SDN techniques in plants, as well
as their possible use by breeding companies to develop new
commercial plants.
3
TIBTEC-1063; No. of Pages 9
Review
SDN
Chromosome
No donor
DNA
NHEJ
Donor DNA
Ligate
Random repair with gain
or loss of base pairs
SDN1
Gene off
Figure 1. Different site-directed nuclease (SDN) techniques (SDN-1, 2, and 3). An SDN complex is shown at the top in association with the target sequence. The repair can
take place via nonhomologous end-joining (NHEJ) or homologous recombination (HR) using the donor DNA. SDN-1 can result in site-specific random mutations by NHEJ.
In SDN-2, a homologous donor DNA is used to induce specific nucleotide sequence changes by HR. In SDN-3 DNA is integrated in the plant genome via HR.
SDN-1 approaches using ZFNs have been used in Ara-
bidopsis, tobacco, petunia, and soybean, whereas TALENs
have been used in Arabidopsis, tobacco, and rice, and MNs
in maize [16,18]. The first application of ZFN enzymes in
plants used a reporter sequence newly incorporated into
the plant genome to screen for ZFN-derived mutants [16].
Subsequently, several articles have reported site-specific
mutations using ZFN constructs stably integrated into the
plant genome [19–23]. Although in some cases short dele-
tions accounted for 80% of the ZFN-induced mutations
[24], in others nucleotide substitutions made up 70% of
all mutations induced [19]. The first ZFN-mediated gene
knockouts in an endogenous target were of the tobacco
acetolactate synthase gene (ALS) named SuRA [25].
TALENs have been used to induce targeted mutations in
protoplasts from Arabidopsis and tobacco [26,27], and to-
bacco leaves [28]. The use of an engineered MN in plants was
first reported in maize with targeted mutagenesis at the
native liguleless locus [29]. A recent study [30] has revealed
TALEN-mediated mutation of the binding site for the natu-
ral TAL effector AvrXa7 or PthXo3 of Xanthomonas oryzae
pv. oryzae upstream of the rice bacterial blight susceptible
gene Os11N3. The obtained mutants did not induce the
Os11N3 in response to the TAL effectors and were resistant
to the pathogen. The plants were morphologically identical
to the wild-type, suggesting that the mutations did not
interfere with the normal expression of the gene.
SDN-2 techniques have also been used to correct non-
functional reporter genes previously introduced into the
plant [16] but also to modify native genes. For example,
4
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Key:
DSB Binding Nuclease
domain domain
+ Donor
+ Donor
DNA
DNA
HR HR
Donor DNA
Paste Paste
Gene modificaon at DNA inseron
one or more posions SDN3
SDN2 Add gene
Gene edit
TRENDS in Biotechnology
ZFNs [31] and TALENs [32] introduced specific point
mutations in the acetolactate synthase genes (als SuRA
and SuRB) to confer resistance to imidazolinone and sul-
fonylurea herbicides.
The first evidence that an SDN-induced break increases
the frequency of targeting was obtained in tobacco using an
MN [16]. More recently the use of SDNs (MNs and ZFNs) to
facilitate the targeted integration of transgenes has been
reported in tobacco [18,33], maize [34], and Arabidopsis
[20]. In all three cases the transgenes were targeted to an
artificial locus previously integrated in the genome.
Targeted gene insertion into a natural chromosomal
target site has only been reported in maize [35] and
tobacco [33]. In the latter, the transgene was targeted to
the stress-related CHN50 endochitinase gene, which is
highly expressed in the stationary phase of suspension
culture cells, and could therefore be particularly suitable
for recombinant protein production.
In addition to the examples reported above, the SDN
technique is being used by plant breeding companies to
introduce new traits in crops. Companies are using SDNs
(in particular MNs and ZFNs) in maize, oilseed rape, and
tomato from the research phase through to phase 3 of
commercial development [9]. Although the companies do
not disclose the traits involved, the techniques are clearly
considered advantageous for accelerated breeding of new
phenotypes [9].
There are many traits that could be improved using SDNs
(Box 3). For targeted mutagenesis (SDN-1 and SDN-2) these
could include: herbicide tolerance (via modifications to the
TIBTEC-1063; No. of Pages 9
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Box 3. Potential uses of SDNs
Potential uses are inumerable. DNA shuffling can generate
sequence variation producing proteins with desirable properties
(kinetics, substrate specificity, temperature or pH optima, and ligand
binding) including improved disease resistance (e.g., R gene
shuffling [68]). SDNs can target the most effective changes required.
Furthermore, different techniques such as TILLING and EcoTILLING
and next-generation sequencing (NGS)/whole genome sequence
analysis are identifying candidate genes and sequences polymorph-
isms that can be used in SDN applications for targeted phenotype
development. Examples [69] include:
(i) virus resistance (targeting translation initiation factors [36]);
(ii) herbicide tolerance (target – acetolactate synthase gene [70]);
(iii) lowering antinutritional compounds (erucic acid in Brassicas –
targeting fatty acid elongases) and allergens (target – con-
glutin genes in peanut) [71,72];
(iv) improved nutritional value via elevated carotenoids, modified
carotenoid balance (target – zeaxanthin epoxidase) [73];
(v) modified starches and fats for food and non-food uses
(targeting starch synthases, branching enzymes, and fatty acid
desaturases) [74,75];
(vi) longer shelf life/reduced wastage [targets include aminocyclo-
propane (ACC) oxidase and polygalacturoanse] [76];
(vii) improved quality by reducing enzymic (target polyphenol
oxidases) and nonenzymic browning (high quality and low
acrylamide potato – target invertase genes) [77];
(viii) yield benefits via modified RuBisCO genes, increasing catalytic
activity and/or decreasing oxygenation activity and improved
seed set (e.g., producing six row barley by targeting home-
odomain leucine zipper genes) [78,79];
(ix) improved biomass conversion for biofuels (lower lignin-target
caffeic acid O-methyltransferase gene) [80].
endogenous EPSPS, ALS, AHAS, or IPK genes), changed
composition (e.g., reduced levels of allergens or phytic acids,
to improve the nutritional value of animal feed, modified
starch for industrial use or to lower glycaemic index), or
resistance to biotic or abiotic stresses. With respect to the
latter, targeted mutagenesis is envisaged for virus resis-
tance via modification of the eiF4E gene in tomato [36],
nematode resistance via modification of the Gro1-4 gene in
potato [37], and abiotic cold stress tolerance via modification
of the DREB1 genes in rice [38,39]. This should encourage an
increased focus on SDN approaches. The targeted insertion
of transgenes (SDN-3) facilitates more predictable expres-
sion (targeting to high expression regions or decreasing the
risk of gene silencing) while minimising impacts on the
genome (avoiding insertion into genes or regulatory
regions). Moreover, SDN-3 may be used for stacking trans-
genes at the same locus, allowing their mobilisation into
other germplasm by crossing as a single locus [18].
Potential safety issues associated with SDN-based
approaches compared with other plant breeding
platforms
SDNs can be used to introduce random or predefined
mutations at specific loci (SDN-1 or SDN-2) or to target
transgenes to a predefined locus (SDN-3). The SDN-based
techniques are thus related to physical or chemical muta-
genesis techniques used in conventional breeding (SDN-1
and SDN-2) and to transgenesis (SDN-3). Therefore, when
analysing the safety of SDN-derived products, a compari-
son with risks associated with other techniques currently
used to produce commercial crops (e.g., conventional mu-
tagenesis and transgenesis) is highly relevant.
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Mutations are key drivers of genome variability and
evolution and therefore important in plant breeding. Spon-
taneous mutations accumulate naturally due to, for exam-
ple, replication errors, movement of transposons and DNA
repair following exposure to UV light (Box 4). Plant bree-
ders also generate new variability using mutagenesis
techniques that are based on the fact that the repair of
most of the DNA damage induced chemically (e.g., via ethyl
methyl sulfonate; EMS) or physically (e.g., via irradiation)
also relies on the cellular repair mechanisms, including the
NHEJ pathway, that can be error prone [40] (Box 2). This
often results in mutations at the repair locus. Mutagenesis
has been widely used by breeders since the second half of
the last century and in 2012 the FAO/IAEA mutant variety
database listed 3200 officially released cultivars in over
200 plant species (http://mvgs.iaea.org). After randomly
mutagenising the plant genomes, forward genetic screens
can select mutants with improved traits for inclusion in
breeding programmes. As the induced mutagenesis is
random, many unwanted mutations occur. However (and
when not limited by the reproductive biology of the spe-
cies), these mutations can be segregated away during the
selection and breeding processes.
The DNA lesions induced by SDN techniques will be
repaired by one of the DNA repair pathways of the cell (Box
2), as would be the case in those exploited by conventional
breeding (spontaneous or induced mutations). Therefore,
the resulting mutations will be indistinguishable. Al-
though designed to cleave DNA only at predefined posi-
tions, SDNs may also cleave DNA at unintended locations
with a certain probability. Off-target activity of the SDN
depends on the specificity of the SDN and the frequency of
sequences similar to the SDN recognition site in the ge-
nome. The off-target cleavage is the main reason for cellu-
lar toxicity observed with ZFNs [6–8]. Different strategies
are followed to decrease off-target mutations including the
use of obligate heterodimers [41,42], cleavage domains of
other restriction enzymes [43], or DNA nickases fused to
the specific DNA-binding domains of SDNs instead of the
currently used restriction enzymes [44,45]. In any case, the
frequency of off-target mutations caused by SDNs would be
well below the frequency of unwanted mutations resulting
from chemical or physical mutagenesis agents, for exam-
ple, one mutation per 150 kbp can be effected by EMS
treatment [46]. This reduces the possibility of unintended
effects potentially having an impact on the safety of the
final product. Furthermore, as in conventional breeding,
unintended mutations can be segregated away during the
selection and breeding process.
The predictability of the outcome of SDN techniques is
even higher in the case of SDN-2, because the mutations to
be introduced in the target locus are predefined by the
sequence of the DNA used as a repair donor.
In contrast with the mutagenic techniques historically
used in plant breeding, SDN-based mutagenesis usually
requires the introduction of the genes encoding the SDNs
by transgenesis. However, the transgene can be segregated
away during the selection and breeding processes because
its presence is not required for the maintenance of the
improved trait. Moreover, SDNs can also be delivered
transiently, avoiding the integration of any transgene
5
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Box 4. DNA damage
Cells are constantly exposed to environmental agents and endogen-
ous processes that inflict damage to DNA (Figure I). Six main
pathways coexist to deal with DNA damage [81]. Three excision
repair pathways exist to cope with DNA single strand breaks: (i) base
excision repair (BER) will repair single-strand breaks, base modifica-
tions, and abasic sites; (ii) nucleotide excision repair (NER) will repair
bulky DNA adducts (pyrimidine dimer); and (iii) DNA mismatch repair
(MMR) will repair mismatch and base modifications. DNA DSBs are
Damage origin
Endogenous damage:
• Replicaon • Transposable • Reacve oxygen
errors elements species
DNA damage
Mismatch and base Inserons, Base modificaons,
modificaons, deleons, abasic sites, DNA
inserons, deleons, DNA breaks breaks
DNA breaks
Figure I. Sources of DNA damage. DNA, our genetic fingerprint, is constantly subjected to mechanisms that can cause damage. These may be driven endogenously
(e.g., via errors in DNA replication, transposable element movement, or oxidative stress resulting from metabolic processes) or exogenously via factors such as
radiation or chemical agents that result in mutations or damage to nucleotide bases, DNA crosslinking, single-strand breaks (SSBs) and double-strand breaks (DSBs).
[16], or directly as proteins [47]. An interesting approach is
the use of virus vectors to transiently deliver the SDNs, as
demonstrated with petunia using the tobacco rattle virus
as a vector to deliver a ZFN that allowed ZFN-mediated
mutations to be recovered in the progeny without requiring
a transgenic step [48].
In addition to introducing mutations at specific loci,
SDNs can be used to introduce long stretches of DNA (e.g.,
transgenes) into the genome (the SDN-3 technique). In
these cases it is pertinent to compare the process and
outcomes with transgenesis, because the aim and the final
products will be similar. However, although in transgen-
esis the DNA is integrated into natural DNA breaks that
occur at random positions in the genome (often close to
actively transcribed genes [49]), SDN-3 techniques target
DNA insertion to a predefined site in the genome. Target-
ing of the transgene in a predefined locus can optimise the
genomic environment for gene expression and minimise
hazards associated with the disruption of genes and/or
regulatory elements in the recipient genome. Moreover,
because the junctions between the transgene and the
recipient genome are predefined by the sequence of the
donor used, the SDN-3 approach should avoid the creation
of new open reading frames with similarity to toxins or
allergens. Therefore, the use of the SDN-3 technique
could be seen as a way to decrease substantially these
perceived risks.
Regulatory considerations
As already stated, SDN techniques can have multiple
advantages for plant breeding. SDN-1 and SDN-2
approaches can create new gene variants with an unprec-
edented specificity and predictability, and SDN-3
6
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
handled by three DNA repair pathways: HR, C-EJ, and A-EJ, also
known as microhomology mediated end joining (MMEJ) [82]. C-EJ
and A-EJ can be grouped under the denomination of NHEJ.
Conventional breeding performed by mutagenesis based on EMS
or ionising irradiation will produce multiple base modifications or
DSBs that can lead to major chromosomal aberrations, chromosome
losses, and large deletions. By contrast, SDNs will produce mostly
targeted DSBs.
Exogenous damage:
• Enzymes • Chemical • Ionizing and UV
(SDN) agents radiaon
DNA double Base modificaons Pyrimidine dimer (e.g., UV),
strand breaks (e.g., EMS), DNA base modificaons, abasic
crosslinks (e.g., sites, DNA breaks,
cisplan), DNA breaks translocaons, large deleons
(e.g., bleomycin) (e.g.,fast neutron, γ- or X-rays)
TRENDS in Biotechnology
approaches can make the production of GMPs more pre-
dictable and could be of great interest for the development
of stacked events. Academic laboratories, consortia, and
specialised biotechnology service providers are improving
the design of SDNs and the methods used to deliver them
into the cell to make them more efficient. Plant breeding
companies have started to use these techniques to develop
a new generation of crops with new traits. However, there
are species-specific limitations. The design of the SDN
targets requires knowledge of the genome sequence, which
is still lacking for some important crops (and certainly for
‘orphan’ crops). Also, the delivery of the SDNs often
requires efficient transformation and regeneration proto-
cols that are not available for all plants [18,50]. Important-
ly, the future of these techniques is in the balance as
uncertainty remains over the legal status of plants gener-
ated by SDN-based approaches and the regulatory over-
sight required. For GMPs, it can currently take almost 6
years and $35 million to generate the data for a regulatory
dossier [9], limiting the use of this technology to high-value
crops and traits developed by the major agrobiotechnology
companies. Thus, finding the appropriate level of regula-
tory oversight to ensure the safe use of these new techni-
ques while keeping development costs at a minimum is of
great importance.
In the past few years several opinion articles have been
published discussing the possible legal status of SDN tech-
niques [2,46,47]. It appears that there is growing agreement
that for SDN-1 techniques that introduce SDNs as proteins
or RNAs (no transgene is involved) the derived products
should be considered for exclusion from genetically modified
organism (GMO) legislation. In the EU, this is also the view
of several competent authorities of individual member
TIBTEC-1063; No. of Pages 9
Review
states, such as the German ZKBS (http://www.bvl.bund.
de/SharedDocs/Downloads/06_Gentechnik/ZKBS/02_
Allgemeine_Stellungnahmen_englisch/01_general_
subjects/zkbs_general_ZFN_1.pdf?__blob=publicationFile&
v=3) and COGEM from The Netherlands (http://www.co-
gem.net/index.cfm/en/publications/publicatie/zinc-finger-
on-the-pulse-developments-and-implications-of-zinc-
finger-technology). Similar opinions were voiced by most
participants at a European Joint Research Centre meet-
ing in 2011, which included representation from
Argentina, Australia, Canada, Japan, the EU, and South
Africa [2]. In the USA, the Department of Agriculture
has informed crop companies that plants obtained using
SDN-1 techniques, together with those in which any
transgene coding for the SDN has been removed by segre-
gation, will not be regulated (http://www.aphis.usda.gov/
biotechnology/reg_loi.shtml) [51].
With respect to plants obtained by SDN-2 and SDN-3 the
US Department of Agriculture has intimated a case-by-case
approach [51]; an approach preferred by some countries
(e.g., Argentina) for SDN-2 [9]. In the EU, some competent
authorities (e.g., the German ZKBS), as well as a majority of
the members of the New Techniques Working Group con-
sidered that SDN-2-derived products should also be consid-
ered for exclusion from the EU Directive that regulates
GMOs when the changes introduced are small and where
no stably introduced transgene is involved in the process
(http://www.bvl.bund.de/SharedDocs/Downloads/06_Gen-
technik/ZKBS/02_Allgemeine_Stellungnahmen_englisch/
05_plants/zkbs_plants_new_plant_breeding_techniques.
pdf?__blob=publicationFile&v=2). For SDN-3, most of the
countries seem to consider plants derived using this tech-
nique to be GMOs. In Europe, the European Food Safety
Authority (EFSA) has issued an opinion on SDN-3, indicat-
ing that although plants obtained by SDN-3 techniques are
similar to GMOs with respect to the introduced trait, the
targeted nature of SDN-3 may minimise the hazards linked
to the insertion of the transgene into the genome [52].
If products derived from SDN techniques are indeed
designated as GMPs, a specific detection method is legally
required to identify the products unambiguously. For most
SDN-3 products this should be relatively easy, because the
newly introduced DNA is relatively long, but may be
extremely challenging for some SDN-1 and SDN-2 pro-
ducts that may differ in only a few nucleotides. Even when
a method could be developed for detection of introduced
mutations, it will be impossible to discriminate them from
the same mutations obtained by conventional mutagenesis
approaches [2,9].
Concluding remarks and outlook
SDN techniques are already proven approaches for site-
specific mutagenesis and targeted DNA insertion in
plants. The specificity they offer holds real promise for
the development, with predictable outcomes, of a new
generation of improved crop varieties with a minimised
potential for unintended effects that could have an impact
on safety. However, some technical limitations as well as
regulatory uncertainty may challenge SDN use for com-
mercial plant breeding. The main technical limitations are
related to both the genomic information needed for the
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
design of the SDNs, which is not available for all crops, and
the existence of an efficient delivery method, which is also
limited to specific genotypes, even within the same spe-
cies. However, information on plant genomes is growing
very rapidly and there is active research in new delivery
methods for SDNs. Indeed the field is developing at a fast
pace and new methods developed in the animal field, such
as the CRISPR approach, might in the future also be used
to improve crop plants. With respect to the regulatory
classification of the products obtained by SDN techniques,
consensus among the different regulatory bodies and sta-
keholders has not been reached yet, in particular, in the
EU. For SDN-3 approaches most regulatory bodies seem to
view them as improved GMOs, whereas products devel-
oped using SDN-1 may, in the future, be considered as non-
GMOs for legislative purposes and particularly when no
transgene is involved. However, absolute clarity and con-
sensus is proving elusive at present with SDN-2 a partic-
ular case in point, because it may fall somewhere between
the two extremes of SDN-1 and SDN-3. There is an urgent
need for a discussion, based on scientific data as much as
possible, on the regulatory framework for these techni-
ques that may clarify their future and allow the invest-
ment the development of these promising techniques
needs.
Acknowledgements
The authors want to thank the members of the EFSA New Techniques
Working Group for seminal discussions at the origin of this manuscript,
and Elisabeth Waigmann for critical reading of the manuscript.
Disclaimer statement
Josep Casacuberta, Howard Davies, and Fabien Nogué are currently ad
hoc members of the Molecular Characterisation Working Group of EFSA
GMO Risk Assessment Panel. Nancy Podevin was, until recently a
member of the unit supporting the EFSA GMO Panel. All of the authors
were members or supported a working group set up by EFSA to develop
the EFSA GMO Panels final opinion on SDN–3 following a mandate from
the European Commission. The opinion is published: [The EFSA GMO
Panel. (2012) Scientific opinion addressing the safety assessment of
plants developed using zinc finger nuclease 3 and other site-directed
nucleases with similar function. EFSA J. 10, 2943 (http://dx.doi.org/
10.2903/j.efsa.2012.2943)]
The authors do not believe these involvements conflict with the paper to
be published in Trends in Biotechnology. The opinions in the manuscript
are those of the authors and not of the EFSA.
References
1 Stamp, P. and Visser, R. (2012) The twenty-first century, the century of
plant breeding. Euphytica 186, 585–591
2 Lusser, M. and Rodrı́guez-Cerezo, E. (2012) Comparative regulatory
approaches for new plant breeding techniques. Workshop proceedings.
European Commission. JRC Technical Report EUR 25237 EN
3 DeFrancesco, L. (2011) Move over ZFNs. Nat. Biotechnol. 29, 681–684
4 Taylor, G.K. et al. (2012) LAHEDES: the LAGLIDADG homing
endonuclease database and engineering server. Nucleic Acids Res.
40, W110–W116
5 Mussolino, C. et al. (2011) A novel TALE nuclease scaffold enables high
genome editing activity in combination with low toxicity. Nucleic Acids
Res. 39, 9283–9293
6 Cornu, T.I. et al. (2008) DNA-binding specificity is a major determinant of
the activity and toxicity of zinc-finger nucleases. Mol. Ther. 16, 352–358
7 Pattanayak, V. et al. (2011) Revealing off-target cleavage specificities of
zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770
8 Gabriel, R. et al. (2011) Genome-wide determination of double-strand
breaks reveals high specificity of zinc finger nucleases. Hum. Gene
Ther. 21, 1371
7
TIBTEC-1063; No. of Pages 9
Review
9 Lusser, M. et al. (2012) Deployment of new biotechnologies in plant
breeding. Nat. Biotechnol. 30, 231–239
10 Roth, N. et al. (2012) The requirement for recombination factors differs
considerably between different pathways of homologous double-strand
break repair in somatic plant cells. Plant J. 72, 781–790
11 Waterworth, W.M. et al. (2011) Repairing breaks in the plant genome:
the importance of keeping it together. New Phytol. 192, 805–822
12 Petolino, J.F. et al. (2010) Zinc finger nuclease-mediated transgene
deletion. Plant Mol. Biol. 73, 617–628
13 Sollu, C. et al. (2010) Autonomous zinc-finger nuclease pairs for
targeted chromosomal deletion. Nucleic Acids Res. 38, 8269–8276
14 Lee, H.J. et al. (2012) Targeted chromosomal duplications and
inversions in the human genome using zinc finger nucleases.
Genome Res. 22, 539–548
15 Fauser, F. et al. (2012) In planta gene targeting. Proc. Natl. Acad. Sci.
U.S.A. 109, 7535–7540
16 Tzfira, T. et al. (2012) Genome modifications in plant cells by custom-
made restriction enzymes. Plant Biotechnol. J. 10, 373–389
17 Parisi, C. et al. (2013) Analysing patent landscapes in plant
biotechnology and new plant breeding techniques. Transgenic Res. 22,
15–29
18 Curtin, S.J. et al. (2012) Genome engineering of crops with designer
nucleases. Plant Genome 5, 42–50
19 Osakabe, K. et al. (2010) Site-directed mutagenesis in Arabidopsis
using custom-designed zinc finger nucleases. Proc. Natl. Acad. Sci.
U.S.A. 107, 12034–12039
20 de Pater, S. et al. (2009) UN-induced mutagenesis and gene-targeting
in Arabidopsis through Agrobacterium-mediated floral dip
transformation. Plant Biotechnol. J. 7, 821–835
21 Even-Faitelson, L. et al. (2011) Localized egg-cell expression of effector
proteins for targeted modification of the Arabidopsis genome. Plant J.
68, 929–937
22 Tovkach, A. et al. (2009) A toolbox and procedural notes for
characterizing novel zinc finger nucleases for genome editing in
plant cells. Plant J. 57, 747–757
23 Zhang, F. et al. (2010) High frequency targeted mutagenesis in
Arabidopsis thaliana using zinc finger nucleases. Proc. Natl. Acad.
Sci. U.S.A. 107, 12028–12033
24 Lloyd, A. et al. (2005) Targeted mutagenesis using zinc-finger
nucleases in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 102, 2232–2237
25 Maeder, M.L. et al. (2008) Rapid ‘‘Open-Source’’ engineering of
customized zinc-finger nucleases for highly efficient gene modification.
Mol. Cell 31, 294–301
26 Cermak, T. et al. (2011) Efficient design and assembly of custom
TALEN and other TAL effector-based constructs for DNA targeting.
Nucleic Acids Res. 39, e82
27 Zhang, Y. et al. (2013) TALENs enable efficient plant genome
engineering. Plant Physiol. 161, 20–27
28 Mahfouz, M.M. et al. (2011) De novo-engineered transcription
activator-like effector (TALE) hybrid nuclease with novel DNA
binding specificity creates double-strand breaks. Proc. Natl. Acad.
Sci. U.S.A. 108, 2623–2628
29 Gao, H.R. et al. (2010) Heritable targeted mutagenesis in maize using a
designed endonuclease. Plant J. 61, 176–187
30 Li, T. et al. (2012) High-efficiency TALEN-based gene editing produces
disease-resistant rice. Nat. Biotechnol. 30, 390–392
31 Townsend, J.A. et al. (2009) High-frequency modification of plant genes
using engineered zinc-finger nucleases. Nature 459, 442–445
32 Zhang, Y. et al. (2012) TALENs enable efficient plant genome
engineering. Plant Physiol. 161, 20–27
33 Cai, C.Q. et al. (2009) Targeted transgene integration in plant cells
using designed zinc finger nucleases. Plant Mol. Biol. 69, 699–709
34 D’Halluin, K. et al. (2008) Homologous recombination: a basis for
targeted genome optimization in crop species such as maize. Plant
Biotechnol. J. 6, 93–102
35 Shukla, V.K. et al. (2009) Precise genome modification in the crop
species Zea mays using zinc-finger nucleases. Nature 459, 437–441
36 Piron, F. et al. (2010) An induced mutation in tomato eIF4E leads to
immunity to two potyviruses. PLoS ONE 5, e11313
37 Milczarek, D. et al. (2011) Suitability of molecular markers for selection
of potatoes resistant to Globodera spp. Am. J. Potato Res. 88, 245–255
38 Tondelli, A. et al. (2011) Inside the CBF locus in Poaceae. Plant Sci.
180, 39–45
8
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
39 Mao, D. and Chen, C. (2012) Colinearity and similar expression pattern
of rice DREB1s reveal their functional conservation in the cold-
responsive pathway. PLoS ONE 7, e47275
40 Tuteja, N. et al. (2009) Genotoxic stress in plants: shedding light on DNA
damage, repair and DNA repair helicases. Mutat. Res. 681, 134–149
41 Doyon, Y. et al. (2011) Enhancing zinc-finger-nuclease activity with
improved obligate heterodimeric architectures. Nat. Methods 8, 74–79
42 Miller, J.C. et al. (2007) An improved zinc-finger nuclease architecture
for highly specific genome editing. Nat. Biotechnol. 25, 778–785
43 Fonfara, I. et al. (2012) Creating highly specific nucleases by fusion of
active restriction endonucleases and catalytically inactive homing
endonucleases. Nucleic Acids Res. 40, 847–860
44 Ramirez, C.L. et al. (2012) Engineered zinc finger nickases induce
homology-directed repair with reduced mutagenic effects. Nucleic
Acids Res. 40, 5560–5568
45 Wang, J. et al. (2012) Targeted gene addition to a predetermined site in
the human genome using a ZFN-based nicking enzyme. Genome Res.
22, 1316–1326
46 Sasaki, T. et al. (2012) Unusual case of apparent hypermutation in
Arabidopsis thaliana. Genetics 192, 1271–1280
47 Gaj, T. et al. (2012) Targeted gene knockout by direct delivery of zinc-
finger nuclease proteins. Nat. Methods 9, 805–807
48 Vainstein, A. et al. (2011) Permanent genome modifications in plant
cells by transient viral vectors. Trends Biotechnol. 29, 363–369
49 Fu, F.F. et al. (2009) Studies on rice seed quality through analysis of a
large-scale T-DNA insertion population. Cell Res. 19, 380–391
50 Barampuram, S. and Zhang, Z. (2011) Recent advances in plant
transformation. In Plant Chromosome Engineering (Birchler, J.,
ed.), pp. 1–35, Humana Press
51 Waltz, E. (2012) Tiptoeing around transgenics. Nat. Biotechnol. 30,
215–217
52 European Food Safety Authority (2012) Scientific opinion addressing
the safety assessment of plants developed using zinc finger nuclease
3 and other site-directed nucleases with similar function. EFSA J.
10, 2943
53 Stoddard, B.L. (2011) Homing endonucleases: from microbial genetic
invaders to reagents for targeted DNA modification. Structure 19, 7–15
54 Hafez, M. and Hausner, G. (2012) Homing endonucleases: DNA
scissors on a mission. Genome 55, 553–569
55 Arnould, S. et al. (2011) The I-CreI meganuclease and its engineered
derivatives: applications from cell modification to gene therapy.
Protein Eng. Des. Sel. 24, 27–31
56 Grizot, S. et al. (2010) Generation of redesigned homing endonucleases
comprising DNA-binding domains derived from two different scaffolds.
Nucleic Acids Res. 38, 2006–2018
57 Carroll, D. (2011) Genome engineering with zinc-finger nucleases.
Genetics 188, 773–782
58 Bogdanove, A.J. et al. (2010) TAL effectors: finding plant genes for
disease and defense. Curr. Opin. Plant Biol. 13, 394–401
59 Boch, J. and Bonas, U. (2010) Xanthomonas AvrBs3 family-type III
effectors: discovery and function. Annu. Rev. Phytopathol. 48, 419–436
60 Boch, J. et al. (2009) Breaking the code of DNA binding specificity of
TAL-type III effectors. Science 326, 1509–1512
61 Moscou, M.J. and Bogdanove, A.J. (2009) A simple cipher governs DNA
recognition by TAL effectors. Science 326, 1501
62 Jinek, M. et al. (2012) A programmable dual-RNA-guided DNA
endonuclease in adaptive bacterial immunity. Science 337, 816–821
63 Cong, L. et al. (2013) Multiplex genome engineering using CRISPR/Cas
systems. Science 339, 819–823
64 Jinek, M. et al. (2013) RNA-programmed genome editing in human
cells. Elife 2, e00471
65 Mali, P. et al. (2013) RNA-guided human genome engineering via Cas9.
Science 339, 823–826
66 Grabarz, A. et al. (2012) Initiation of DNA double strand break repair:
signaling and single-stranded resection dictate the choice between
homologous recombination, non-homologous end-joining and
alternative end-joining. Am. J. Cancer Res. 2, 249–268
67 Nimonkar, A.V. et al. (2011) BLM-DNA2-RPA-MRN and EXO1-BLM-
RPA-MRN constitute two DNA end resection machineries for human
DNA break repair. Genes Dev. 25, 350–362
68 Wulff, B.B. et al. (2001) Domain swapping and gene shuffling identify
sequences required for induction of an Avr-dependent hypersensitive
response by the tomato Cf-4 and Cf-9 proteins. Plant Cell 13, 255–272
TIBTEC-1063; No. of Pages 9
Review
69 Kurowska, M. et al. (2011) TILLING: a shortcut in functional genomics.
J. Appl. Genet. 52, 371–390
70 Shimizu, M. et al. (2011) Application of mutated acetolactate synthase
genes to herbicide resistance and plant improvement. In Herbicides,
Theory and Applications (Larramendy, M., ed.), InTech
71 Tian, B.M. et al. (2011) Decreasing erucic acid level by RNAi-mediated
silencing of fatty acid elongase 1 (BnFAE1.1) in rapeseeds (Brassica
napus L.). Afr. J. Biotechnol. 10, 13194–13201
72 Foley, R.C. et al. (2011) Identification and characterisation of seed
storage protein transcripts from Lupinus angustifolius. BMC Plant
Biol. 11, 59
73 Yang, Q.J. et al. (2012) Functional characterization of the Gentiana
lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato
plants. Transgenic Res. 21, 1043–1056
74 Crofts, N. et al. (2012) Lack of starch synthase IIIa and high expression
of granule-bound starch synthase I synergistically increase the
apparent amylose content in rice endosperm. Plant Sci. 193, 62–69
75 Chi, X.Y. et al. (2011) Genome-wide analysis of fatty acid desaturases in
soybean (Glycine max). Plant Mol. Biol. Rep. 29, 769–783
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
76 Yun, S.S. et al. (2011) Isolation of fruit ripening genes from Carica
papaya var. Eksotika I cDNA libraries. J. Trop. Agric. Food Sci. 39,
203–211
77 Toevs, E. et al. (2011) An industry perspective of all-native and
transgenic potatoes. AgBioForum 14, 14–19
78 Whitney, S.M. et al. (2011) Advancing our understanding and capacity
to engineer nature’s CO2-sequestering enzyme, Rubisco. Plant Physiol.
155, 27–35
79 Sakuma, S. et al. (2010) Duplication of a well-conserved
homeodomain-leucine zipper transcription factor gene in barley
generates a copy with more specific functions. Funct. Integr.
Genomics 10, 123–133
80 Fu, C.X. et al. (2011) Genetic manipulation of lignin reduces
recalcitrance and improves ethanol production from switchgrass.
Proc. Natl. Acad. Sci. U.S.A. 108, 3803–3808
81 Ciccia, A. and Elledge, S.J. (2010) The DNA damage response: making
it safe to play with knives. Mol. Cell 40, 179–204
82 Symington, L.S. and Gautier, J. (2011) Double-strand break end
resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271