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REVIEW
Progress of targeted genome modification approaches in higherplants
Teodoro Cardi1 • C. Neal Stewart Jr.2
Received: 11 February 2016 / Accepted: 21 March 2016 / Published online: 29 March 2016
� Springer-Verlag Berlin Heidelberg 2016
Abstract Transgene integration in plants is based on
illegitimate recombination between non-homologous
sequences. The low control of integration site and number
of (trans/cis)gene copies might have negative conse-
quences on the expression of transferred genes and their
insertion within endogenous coding sequences. The first
experiments conducted to use precise homologous recom-
bination for gene integration commenced soon after the
first demonstration that transgenic plants could be pro-
duced. Modern transgene targeting categories used in plant
biology are: (a) homologous recombination-dependent
gene targeting; (b) recombinase-mediated site-specific
gene integration; (c) oligonucleotide-directed mutagenesis;
(d) nuclease-mediated site-specific genome modifications.
New tools enable precise gene replacement or stacking
with exogenous sequences and targeted mutagenesis of
endogeneous sequences. The possibility to engineer chi-
meric designer nucleases, which are able to target virtually
any genomic site, and use them for inducing double-strand
breaks in host DNA create new opportunities for both
applied plant breeding and functional genomics. CRISPR is
the most recent technology available for precise genome
editing. Its rapid adoption in biological research is based on
its inherent simplicity and efficacy. Its utilization, however,
depends on available sequence information, especially for
genome-wide analysis. We will review the approaches used
for genome modification, specifically those for affecting
gene integration and modification in higher plants. For each
approach, the advantages and limitations will be noted. We
also will speculate on how their actual commercial devel-
opment and implementation in plant breeding will be
affected by governmental regulations.
Keywords Homologous recombination � Recombinases �Oligonucleotide-directed mutagenesis � Nucleases �Targeted mutagenesis � Site-specific integration
Introduction
In 1983, four independent studies were published on the
stable genetic transformation of higher plants (Bevan et al.
1983; Fraley et al. 1983; Herrera-Estrella et al. 1983; Murai
et al. 1983). In light of conventional breeding technologies
available then, these papers represented a great leap for-
ward in genetic modification technology. The ability to
choose specific genes in plant genomes to modify for
cultivar development revolutionized crop breeding. Sub-
sequently, the general concept was refined to control
transgene expression in space and time; i.e., in specific
tissues, developmental stages and environments. Not only
could new heterologous genes be introduced and expressed
in the plant chassis, but endogenous gene expression could
be downregulated using antisense or RNAi technologies
(Koch and Kogel 2014; Sheehy et al. 1988). Moreover,
transgenes could also be integrated into plastid genome
(Svab et al. 1990). More recently, the use of plant
sequences derived from cross-compatible species was
advocated in the so-called ‘‘cisgenic’’ gene transfer, with
Communicated by M. Mahfouz.
& Teodoro Cardi
teodoro.cardi@crea.gov.it
1 Consiglio per la Ricerca in Agricoltura e l’Analisi
dell’Economia Agraria (CREA), Centro di Ricerca per
l’Orticoltura, Via Cavalleggeri 25, 84098 Pontecagnano, Italy
2 Department of Plant Sciences, University of Tennessee,
Knoxville, TN 37996, USA
123
Plant Cell Rep (2016) 35:1401–1416
DOI 10.1007/s00299-016-1975-1
the intention to counteract some of the objections to
‘‘conventional’’ transgenesis (Schouten et al. 2006).
Standard transformation techniques using Agrobac-
terium or biolistics currently do not make all facile trans-
gene targeting into a specific locus. The insertion of genes
into the nuclear plant genome is random and based on non-
homologous end-joining (NHEJ). Therefore, many trans-
genic plant events must be produced to mitigate potential
negative consequences of unpredictable epigenetic modi-
fication of transgene expression, rearranged integration,
interruption of native genes, and vector backbone integra-
tion, i.e., ‘‘position effects’’ (Butaye et al. 2005; Ow 2002).
Precise gene targeting is also desirable in functional stud-
ies. Finally, random integration has negative implications
in the governmental regulation of transgenic plants. Hence,
it is important to develop technologies to target transgene
insertion, which would have importance for both agricul-
ture and basic plant biology research. The end goal is to
select the exact locus for transgene integration and enable
precise transgene landing by homologous recombination
(HR): heretofore, a very challenging quest.
Recent advancements of DNA sequencing technologies
have facilitated an exponential increase of knowledge
about structure and function of plant genomes (Michael
and VanBuren 2015). Such information enabled the
development of several advanced methods for targeted
genome modification in higher plants. Utilizing HR, tech-
nologies include site-specific nucleases to create double
strand breaks, recombinases for gene integration, and
oligonucleotides for precise mutagenesis. We will review
the literature on various genome engineering techniques,
noting, for each approach, major advantages and
limitations.
Homologous recombination-dependent genetargeting
Higher plants, in contrast with several other organisms,
rarely use HR in their nuclear genomes. HR occurs in
somatic cells between 10-3 and 10-6 true gene targeting
(TGT) events in comparison with random integration from
NHEJ, one side invasion (OSI) and ectopic gene targeting
(EGT) (Yamauchi and Iida 2015). On the other hand,
thanks to their prokaryotic origin, HR is a common phe-
nomenon in plastid genomes and is regularly exploited in
transplastomic approaches (Svab et al. 1990). Hence, after
the pioneering experiments of Paszkowski and colleagues
in the late 1980s based on the recovery of antibiotic
resistance after reconstruction of a deleted non-functional
drug-resistance gene in transgenic tobacco plants (Pasz-
kowski et al. 1988), researchers attempted to increase HR
frequency in plant nuclear genomes. Enabling HR in plant
genomes was recognized as an important goal in crop
engineering. Such approaches included schemes based on
gene specific selection (GSS), positive–negative selection
(PNS), and the manipulation of expression of specific
proteins involved in host DNA recombination and repair
(Fig. 1). Gene targeting by any of these approaches can
result in gene knockout/replacement as well as targeted
point mutations. Molecular mechanisms involved in
recombination and integration as well as factors affecting
frequency of HR have been reviewed elsewhere (Da Ines
and White 2013; Iida and Terada 2005; Puchta and Fauser
2013; Yamauchi and Iida 2015).
GSS approaches are generally limited to endogeneous
genes conferring acquired drug resistance after introduc-
tion of point mutations. They have been applied in tobacco,
Arabidopsis thaliana and rice, but TGT was successfully
achieved in just a limited number of cases (Da Ines and
White 2013; Iida and Terada 2005; Yamauchi and Iida
2015). Among these, one Arabidopsis plant out of 750
transformants was obtained after targeting the AGL5
MADS-box regulatory gene using Agrobacterium-medi-
ated floral dip transformation wherein a construct with a
kanamycin-resistance cassette was inserted between 3- and
2-kb homologous segments from the 50 and 30 ends,
respectively, of the target gene (Kempin et al. 1997).
Subsequently, using the same transformation methods, the
protoporphyrinogen oxidase gene (PPO) was targeted to
introduce two mutations conferring herbicide resistance,
but only 3 TGT events were identified, corresponding to a
frequency of 0.72 9 10-3 (Hanin et al. 2001). In rice,
Agrobacterium-mediated transformation was used to
introduce two point mutations in the acetolactate synthase
(ALS) gene, resulting in herbicide resistance. Plants with
only the two desired mutations without any insertion of
foreign DNA were obtained with an estimated gene tar-
geting efficiency of about 1 event in 2000–3000 potential
transformants (Endo et al. 2007). In another report (Saika
et al. 2011), precise mutations in OASA2, which is a key
gene encoding an enzyme in tryptophan (Trp) biosynthesis,
were introduced by Agrobacterium-mediated transforma-
tion with subsequent selection of gene targeted cells using
a Trp analog. Mutant rice plants harboring mutated OASA2
conferred insensitivity to feedback inhibition to Trp and its
analogues when high amounts of tryptophan was accu-
mulated. Nevertheless, in that paper, the authors
acknowledged the need for alternative methods for gene
targeting in rice and other crops.
By contrast with GSS, PNS-based approaches can be
applied, theoretically, to any gene. They rely on positive
(e.g. antibiotic resistance genes) and negative
selectable markers placed within and outside the homolo-
gous sequence fragments, respectively. A conditional
negative selectable marker, the cytosine deaminase gene
1402 Plant Cell Rep (2016) 35:1401–1416
123
(codA) from Escherichia coli, converts non-toxic 5-fluo-
rocytosine (5-FC) into the toxic agent 5-fluorouracil (5-
FU). In contrast, the cell-autonomous non-conditional
negative selection Corynebacterium diphtheriae toxin
fragment A (DT-A) gene confers toxicity only to the cells
in which it is expressed, but not to the adjacent cells
lacking the toxin B fragment (Da Ines and White 2013; Iida
and Terada 2005; Yamauchi and Iida 2015). The first
successful report of PNS-based gene targeting in plants was
achieved in rice in 2002 (Terada et al. 2002). The Waxy
(granule-bound starch synthase) gene was knocked-out
using a targeting vector containing the hpt and the DT-A
genes, and 6.3–6.8 kb homologous flanking regions. Six
plants with TGT were regenerated from 638 hygromycin-
resistant calli obtained with the same vector, and such high
frequency was attributed to the competence of the starting
material, the stringency of the selection regime, as well as
to the efficiency of the PCR screening. Subsequently, using
basically the same protocol, two additional genes (alcohol
dehydrogenase2, Adh2; b1,2-xylosyltransferase, Xyl) wereprecisely targeted and knocked-out at similar frequencies
(Ozawa et al. 2012; Terada et al. 2007). Besides knockout
experiments, knock-in targeting was achieved in rice by the
precise insertion of a promoterless GUS gene 30 of the
promoter of several genes involved in DNA methylation/
demethylation. In contrast with plants expressing GUS
from randomly integrated transgenes, targeted plants had
reproducible and dosage-dependent GUS expression pat-
terns. Furthermore, since they had also the endogenous
target genes knocked-out, they could be used to assess the
function of promoters and encoded enzymes in different
phases of plant growth (Moritoh et al. 2012; Ono et al.
2012; Yamauchi, et al. 2009, 2014). Finally, the PNS
strategy was also used to introduce indels or point
mutations at a targeted locus allowing functional studies of
specific domains or regulatory sequences. In rice, mis-
sense mutations were introduced in the IRE1 gene to pro-
duce plants defective in kinase or RNase activities (Wakasa
et al. 2012), or in the OsRac1 gene to constitutively syn-
thesize an active form of the protein to affect cell responses
to pathogens (Dang et al. 2013). In the latter report, using
the Cre-lox recombination system, the hpt marker gene
could be removed in gene targeted plants, which resulted in
conferring only the induced mutation in the coding
sequence of the gene and a single loxP site in one of its
introns. Cre-lox was also used to remove the hpt gene from
the Waxy gene, allowing its reactivation (Terada et al.
2010). To facilitate high frequency of marker gene removal
and avoid the presence of residual recognition sequences in
targeted plants, the use of a transposase or of an engineered
endonuclease has been discussed (Yamauchi and Iida
2015). The piggyBac-mediated marker excision system
was applied to remove the hpt marker gene from the tar-
geted ALS locus in which two herbicide resistance muta-
tions had been introduced. More than 90 % of regenerated
plants contained two point mutations in the ALS gene and
lacked the piggyBac transposon carrying the hpt gene
(Nishizawa-Yokoi et al. 2015a). With the aim to establish a
universally applicable positive–negative gene targeting
system in plants, a selection system based on both sense
and antisense neomycin phosphotransferase II (nptII) was
designed. Thus far it has been used to knock out the
endogenous Waxy and 33-kD globulin rice genes (Nishi-
zawa-Yokoi et al. 2015b). In contrast to the success in rice,
PNS-based approaches have failed in Arabidopsis and
Lotus japonicus (Gallego et al. 1999; Thykjaer et al. 1997;
Xiaohui Wang et al. 2001). Besides considerations on the
competence of cells used for transformation or general
Fig. 1 Homologous recombination-dependent gene targeting. a In
GSS (gene specific selection) schemes, an endogeneous target gene is
replaced by a copy of the same gene carrying a mutation that can be
selected for. b In PNS (positive–negative selection) schemes,
selection of recombinant products derived by homologous
recombination relies on positive (e.g. antibiotic resistance genes)
and negative selection markers (codA, DT-A genes) placed within and
outside the homologous sequence fragments, respectively. Because of
flanking negative selection markers (striped boxes), products of
random integration are selected against
Plant Cell Rep (2016) 35:1401–1416 1403
123
efficiency of the transformation system, in all latter reports
the less efficient codA negative selection system (relative to
DT-A) was used in rice. When an improved codA variant
was used, the caffeic acid O-methyltransferase (CAOMT)
gene in rice was targeted with efficiencies similar to those
achieved with DT-A (Osakabe et al. 2014).
Increasing the frequency of HR in higher plants has been
attempted by modifying the synthesis of proteins involved
in recombination and chromatin remodeling. However,
only the overexpression of the yeast RAD54 gene, a
member of SWI2/SNF2 gene family involved in chromatin
remodeling, promoted HR, which resulted in increasing
gene targeting by one to two orders of magnitude in Ara-
bidopsis (Shaked et al. 2005). All other attempts resulted in
no improvement of gene targeting efficiency (reviewed in
Da Ines and White 2013).
Recombinase-mediated site-specific geneintegration
Recombinases are common in prokaryotes and lower
eukaryotes, in which they participate in various biological
functions. One example is phage integration into the host
genome of bacteria, in which an enzyme interacts with
specific target sequences and induces their site-specific
recombination. Based on the active amino acid within the
catalytic domain and other features, available systems are
divided into families and subfamilies: (a) bidirectional
tyrosine recombinases (e.g. Cre-lox, FLP-FRT, R-RS),
(b) unidirectional tyrosine recombinases (e.g. k-attB/attP),(c) small serine recombinases (e.g. b-six, cd-res, CinH-RS2), (d) large serine recombinases (e.g. phiC31-attB/attP,
Bxb1-attB/attP). Type (a) systems possess identical
recognition sites and are able to control reversible excision
and integration of a given nucleotide sequence, whereas the
other three types are able to control only unidirectional
reactions either because enzymes recognize non-identical
sites [types (b) and (d)] or for topological constraints [type
(c)] (reviewed by (Lyznik et al. 2003; Thomson and Blechl
2015; Wang et al. 2011).
Following pioneering demonstrations of E. coli phage
1–derived Cre-lox functions in unicellular and multicellular
eukaryotes, including plants, almost 30 years ago (Dale
and Ow 1990, 1991; Odell et al. 1990; Russell et al. 1992;
Sauer 1987; Sauer and Henderson 1988), plants have been
targeted for various applications using a number of sys-
tems. Recombinases have been used for marker gene
excision, gene expression switching, resolution of complex
transgene sites, chromosome manipulation, gene integra-
tion, and gene stacking [reviewed in (Ow 2011, 2016;
Srivastava and Gidoni 2010; Srivastava and Thomson
2016; Thomson and Blechl 2015; Wang et al. 2011)].
Recombinase-mediated site-specific transgene integration
(SSI) was attempted using mostly the bi-directional Cre-lox
system (Albert et al. 1995; Kerbach et al. 2005; Louwerse
et al. 2007; Srivastava and Ow 2002; Vergunst and
Hooykaas 1998; Vergunst et al. 1998), although other
bidirectional (R-RS, FLP-FRT) and unidirectional (Bxb1-
attB/attP, phiC31-attB/attP) systems were tested too (De
Paepe et al. 2013; Hou et al. 2014; Li et al. 2009; Nandy
and Srivastava 2011, 2012; Nanto and Ebinuma 2008;
Nanto et al. 2005; Yau et al. 2011). Cre-lox was purported
to precisely insert large DNA fragments (up to 230 kb) for
complementation studies (Choi et al. 2000), while that of
the phiC31 phage integrase to increase plastid transfor-
mation efficiency in species where the plastid homologous
recombination machinery was not effective (Lutz et al.
2004).
SSI by co-integration (Srivastava and Gidoni 2010)
relies on the recombination of two sites in trans, one pre-
viously inserted in the target genome and the other one in
the donor plasmid (Fig. 2a). However, in the case of
reversible bidirectional systems, the integration reaction is
quite unstable and not favored in comparison with excision.
As a consequence, the inserted sequence can be readily
excised from the recombination of the two identical prox-
imal sites. To stabilize the integrated sequence, several
technological improvements were devised. As a first
application in plants, tobacco protoplasts were engineered
with two lox sites that had slightly different sequences that
still had competence for recombination. However, reverse
recombination was not possible after the forward integra-
tion reaction. In addition, the Cre activity on recombined
sites was significantly reduced by its transient expression or
by displacement of its promoter after integration (Albert
et al. 1995). Other systems have used the same concepts to
disallow the integration of the vector backbone (Nandy and
Srivastava 2011; Srivastava and Ow 2002; Vergunst and
Hooykaas 1998; Vergunst et al. 1998).
The recombinase-mediated cassette exchange strategy
(RMCE) relies on a DNA segment that usually contains a
marker gene flanked by two recognition sites in opposite
orientation (to avoid excision), which is inserted randomly
in the target genome (Fig. 2b). Such ‘‘target cassette’’ is
replaced by an ‘‘exchange cassette’’ that contains the
sequences of interest flanked by the same recognition sites
used prior, which is obtained after a double crossing-over
occurs between them. In 2001, its first application in plants
(maize) was reported in a patent by Baszczynski and col-
leagues (cited by (Ow 2002). Subsequently, Agrobac-
terium-mediated transformation and the R-RS
recombination system were used to introduce, in two sub-
sequent steps, the ‘‘target cassette’’ with nptII and codA
genes and the ‘‘exchange cassette’’ with hpt and luc genes
into tobacco cells (Nanto et al. 2005). Beside the
1404 Plant Cell Rep (2016) 35:1401–1416
123
‘‘exchange cassette’’, the R and ipt genes coding, respec-
tively, for the recombinase and isopentenyl transferase,
which catalyses cytokinin biosynthesis, were also present.
After the first transformation, only plants containing a
single copy of the ‘‘target cassette’’ were selected for re-
transformation, while, subsequently, plants with an abnor-
mal phenotype conferred by the ipt gene retained after
random integration were discarded. In such plants, the
‘‘exchange cassette’’ and the two additional genes could be
eliminated by recombination of two RS sites in the same
orientation. Afterwards, based also on parallel develop-
ment in other organisms, the same group further developed
the technology using a negative selection scheme and
producing plants without any marker genes (Ebinuma et al.
2015; Nanto and Ebinuma 2008). The RMCE strategy was
also applied in Arabidopsis, soybean and aspen, using, in
some cases, non-compatible heterospecific lox or FRT
recombination sites to limit unwanted deletion and inver-
sion of the inserted cassettes (Fladung and Becker 2010; Li
et al. 2009; Louwerse et al. 2007).
The combined use of two or more recombinases and
related target sites to allow marker-free site-specific gene
integration in plants, using either a co-integration or a
RMCE strategy, was proposed (Srivastava and Ow 2004).
Later reports used both bidirectional and unidirectional
recombinases in a variety of schemes to produce marker-
free plants in mono- and dicotyledonous species (De Paepe
et al. 2013; Ebinuma et al. 2015; Fladung and Becker 2010;
Nandy and Srivastava 2012; Nanto et al. 2005).
Transgene stacking in a single pre-defined locus is
desirable for concerted transgene expression and for sub-
sequent introgression of multiple genes into commercial
lines, the latter sometimes not being amenable to trans-
formation. Bidirectional and unidirectional recombinase
systems can be used for this purpose. In soybean, using a
FLP-FRT-mediated RMCE approach, three genes involved
in oil biosynthesis, three in essential amino acid biosyn-
thesis and the ALS marker gene were stacked in a single
genomic locus (Li et al. 2010). Expected phenotypes and
Mendelian segregation were observed for all transgenes in
the plants. Following the combined use of a unidirectional
large serine recombinase (either phiC31-att or BxB1-att),
the stacking of multiple genes associated with the removal
of marker genes has been recently demonstrated in Ara-
bidopsis and tobacco (De Paepe et al. 2013; Hou et al.
2014). A novel approach for gene stacking involving Cre-
mediated site-specific integration followed by nuclease-
mediated marker gene excision (using either heat-inducible
I-SceI or a zinc finger nuclease (ZFN)) has been recently
proposed (Nandy et al. 2015).
In plants containing a single transgene copy that were
produced using site-specific integration, we expect
enhanced predictability and consistency of transgene
expression relative to random and multigenic insertion
events. Such a result has been observed in several studies in
rice and tobacco (Chawla et al. 2006; Nandy and Srivas-
tava 2012; Nanto et al. 2009; Srivastava et al. 2004). For
unknown reasons (perhaps epigenetic or the presence of
plasmid backbones), transgene silencing occurred in some
experiments using Cre-lox-mediated transformation of
PEG-treated tobacco protoplasts (Day 2000; Srivastava and
Gidoni 2010). Similarly, some silencing was also observed
in about one-third of regenerated tobacco plants produced
by PEG transformation of tobacco protoplasts using vectors
Fig. 2 Recombinase-mediated site-specific gene integration. a In co-
integration approaches, gene integration depends on a single crossing-
over between recombination sites (triangles). The integration reaction
is favored over excision by the use of mutant recognition sites that are
not able to recombine after integration. In addition, the gene encoding
for the recombinase is displaced by its promoter after the integration
reaction. b In the recombinase-mediated cassette exchange strategy
(RMCE), a ‘‘target cassette’’ flanked by two recombination sites is
replaced by an ‘‘exchange cassette’’, containing the sequences of
interest flanked by the same recognition sites as before, after a double
crossing-over. In both strategies, recombination sites must be
previously inserted in the plant genome by transformation. P pro-
moter, R recombinase, M1 first marker gene, M2* promoterless
second marker gene, Goi gene of interest
Plant Cell Rep (2016) 35:1401–1416 1405
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containing the Bxb1-att and Cre-lox recombinase systems
(Hou et al. 2014). Nanto and colleagues (2009) did not find
any correlation between expression of transgenes and tar-
get genes after RMCE-mediated transformation.
Oligonucleotide-directed mutagenesis
Targeted oligonucleotide-directed mutagenesis (ODM) has
been used in mammalian and plant cells. Various kinds of
molecules have been developed for such a purpose,
including single stranded RNA/DNA hybrids
(chimeraplasts), ssDNA oligonucleotides, triplex-forming
oligos and others. Chimeric RNA/DNA and ssDNA oligos
have been mostly used in plant cells (Fig. 3). The former
are typically 68 and 88 nucleotides in length, and include
self-complementary DNA and RNA-DNA-RNA strands
identical to the target region except for the specific
base(s) to be changed. The addition of two hairpin loops
flanking the complementary strands prevents concatamer-
ization and degradation. ssDNA oligos, usually about 40
nucleotides long, are homologous to the target region and
include one or few mismatch bases in the central part of the
oligo. The 50 and 30 ends can be protected either with
phosphorothioate linkages or with the fluorescent label Cy3
and a reverse cytosine base. After hybridization, mis-
matches are repaired by a gene conversion mechanism,
inducing site-directed mis-sense/nonsense mutations from
base substitutions or frameshift mutations from indels,
either in coding or regulatory target sequences [recently
reviewed in (Da Ines and White 2013; Gocal et al. 2015;
Rivera-Torres and Kmiec 2016; Sauer et al. 2016)].
Applications in plant cells have been few—mainly
limited to modification of marker genes such as gfp and
herbicide resistance genes. RNA/DNA oligos have been
introduced into tobacco, maize, and rice cells by particle
bombardment or protoplast electroporation, in which single
base substitutions that confer targeted herbicide resistance
have been produced (Beetham, et al. 1999; Kochevenko
and Willmitzer 2003; Okuzaki and Toriyama 2004; Zhu
et al. 1999, 2000). In some cases, however, a 50 shift of theinduced mutation was observed with respect to the targeted
base. Transmission through meiosis and Mendelian inher-
itance of induced mutations have been observed in progeny
(Kochevenko and Willmitzer 2003; Zhu et al. 2000).
Recovery of GFP expression was obtained in tobacco,
maize and wheat cells by either induced frameshift muta-
tions or base substitutions (Beetham et al. 1999; Dong et al.
2006; Zhu et al. 1999). In direct comparisons, Cy3-labeled
ssDNA oligos appeared to be more effective than chimeric
RNA/DNA in a transient assay in wheat, but opposite
results were obtained in tobacco (Dong et al. 2006;
Kochevenko and Willmitzer 2003). A gene editing plat-
form for protoplasts using all-DNA oligonucleotides and
PEG-mediated delivery was developed in oilseed rape to
edit herbicide resistance genes and blue/green fluorescence
genes. Mutated herbicide resistant lines produced for
eventual commercialization were obtained in this way
(Gocal et al. 2015).
Despite the positive results reported above, the general
applicability of ODM in plant and animal systems and its
use in functional genomics studies have been questioned
early in the development of methodologies (Feldmann
1999; Ruiter et al. 2003; van der Steege et al. 2001).
Recently, it was shown that the frequency of gene editing
could be increased by the combined use of oligonucleotides
and engineered nucleases in rice, maize and flax (Sauer
et al. 2016; Shan et al. 2013; Svitashev et al. 2015; Wang
et al. 2015a).
Nuclease-mediated site-specific genomemodifications
The demonstration that double strand breaks (DSBs) can be
enzymatically induced in plant cells and that homologous
recombination could be increased by two orders of mag-
nitude (Puchta et al. 1993, 1996) opened the way to the
development of a wide number of targeted genome modi-
fications in higher plants, aiming to site-specific
Fig. 3 Oligonucleotide-directed mutagenesis. a Chimeric DNA/RNA
oligonucleotides (chimeraplasts) consist of two homologous filaments
that are flanked by two hairpins. One filament contains only
deoxyribonucleotides (blue), whereas the other contains both deoxyri-
bonucleotides and modified ribonucleotids (green). b Single-stranded
oligonucleotides consist only of deoxyribonucleotides with a Cy3 dye
at one end and a reverse base at the other. Both oligonucleotide types
contain mismatch regions (red) that induce gene conversion and the
desired mutation during the repair
1406 Plant Cell Rep (2016) 35:1401–1416
123
mutagenesis or site-specific gene integration. DSBs can be
induced by native or engineered homing endonucleases
(meganucleases), or chimeric endonucleases linked either
to protein (ZFNs and TALENs) or RNA sequences
(CRISPR/Cas9) (Fig. 4) [reviewed, among others, by
(Chen and Gao 2014; Da Ines and White 2013; Fichtner
et al. 2014; Lee et al. 2016; Osakabe and Osakabe 2015;
Puchta and Fauser 2013, 2014, 2015; Rinaldo and Ayliffe
2015; Voytas 2013; Weeks et al. 2016) as well as in other
articles of this issue].
Meganucleases are rare cutting enzymes indigenous to
microrganisms. Following the first experiments aiming to
understand mechanisms of transgene integration and DSB
repair in higher plants (Chilton and Que 2003; Puchta
et al. 1993, 1996; Salomon and Puchta 1998; Tzfira et al.
2003), site-specific transgene integration in a pre-engi-
neered I-SceI site was demonstrated in maize (D’Halluin
et al. 2008). However, similarly to recombinases, the
possibility to select an integration locus depends on pre-
vious (random) introduction of the meganuclease recog-
nition site. Therefore, engineered native meganucleases
were produced to recognize novel targets, but since the
cleavage and DNA-binding sites usually overlap, there
were issues in implementation (Daboussi et al. 2015;
Voytas 2013). Nonetheless, engineered I-CreI enzymes
have been used for targeted mutagenesis in maize and
trait stacking in cotton (D’Halluin et al. 2013; Djukanovic
et al. 2013; Gao et al. 2010). In the latter species, two
herbicide resistance genes were integrated by HR close to
two genes that had been previously inserted; the four
genes segregated together in a single locus in Mendelian
fashion (D’Halluin et al. 2013). An efficient I-SceI-based
method for efficient gene targeting in planta was per-
formed in Arabidopsis and recommended for species for
which efficient transformation and regeneration systems
were not available (Fauser et al. 2012; Puchta and Fauser
2013).
ZFNs are chimeric nucleases in which DNA binding and
cleavage properties are conferred by 3-4 modules of zinc
finger proteins fused to the FokI restriction endonuclease,
which enable locus targeting without the pre-insertion of
enzyme recognition sites. After pioneering work with
animal systems ca. 2000, ZFNs were tested first in plants
using Arabidopsis and tobacco as models, then other plant
species [reviewed in (Petolino 2015; Qi 2015; Voytas
2013)]. Targeted ZFN-induced mutagenesis (through the
NHEJ repair pathway) was first achieved in Arabidopsis
targeting a pre-engineered sequence (Lloyd et al. 2005)
and, subsequently, other species (de Pater et al. 2009;
Marton et al. 2010; Peer et al. 2015). Targeted mutagenesis
of endogeneous genes by inducible ZFNs was first reported
in two back-to-back Arabidopsis papers (Osakabe et al.
2010; Zhang et al. 2010). Shortly thereafter, targeted
mutagenesis in one transgene and nine endogeneous genes
was attempted in soybean, showing also transmission of
ZFN-induced mutations in the subsequent generation
(Curtin et al. 2011). Homologous recombination-based
gene targeting, after induction of DSB in pre-engineered
target sites by ZFN, was first accomplished in tobacco
protoplasts and Arabidopsis plants (de Pater et al. 2009;
Wright et al. 2005). In the same period, in maize, the ipk1
gene was replaced by the PAT gene, obtaining at the same
time phytate-less herbicide resistant plants (Shukla et al.
2009). Through a gene editing approach, point mutations
were precisely introduced in specific codons of ALS SuR
genes in tobacco, resulting in herbicide resistant calli
(Townsend et al. 2009). Precise gene editing was demon-
strated in Arabidopsis with the PPO gene, in which two
mutations conferring herbicide resistance could be induced
(de Pater et al. 2013). More recently, a targeted gene
Fig. 4 Nuclease-mediated site-specific genome modifications. After
targeted induction of a double strand break (DSB) by different kinds
of nucleases, DNA is repaired by non homologous end-joining
(NHEJ) in the absence of donor repair DNA (a) or by homology-
directed repair (HDR) in the presence of various kinds of donor
molecules (b, c, d). a Frameshift mutations are usually induced
resulting in gene disruption, whereas b induced point mutations result
in gene editing. Depending where the DSB is formed, gene insertion
will result either in gene replacement (c) or gene stacking (d). ZFNzinc finger nucleases, TALEN transcription activator-like effector
nucleases, CRISPR/Cas Clustered Regularly Interspaced Short Palin-
dromic Repeats/CRISPR-associated systems
Plant Cell Rep (2016) 35:1401–1416 1407
123
exchange mediated by a ZFN double digestion was shown
in tobacco cells (Schneider et al. 2016). ZFN-mediated
DSB formation and HDR (homology directed repair) were
also exploited for trait stacking approaches in maize
(Ainley et al. 2013; Kumar et al. 2015), showing sequential
integration and cosegregation of linked herbicide/insect
resistance genes.
Transcriptional activator-like effectors (TALEs) are
produced by pathogenic plant bacteria (Xanthomonas spp.)
and are involved in pathogenicity. Two TALE genes, each
consisting of 12–30 repeats to encode specific nucleotide
binding capabilities are linked each to a FokI nuclease
gene. TALE nucleases (TALENs) can be designed to rec-
ognize and cleave virtually any genomic locus (Christian
and Voytas 2015; Mahfouz and Li 2011; Sprink et al.
2015).TALENs have been used to genome-edit a wide
number of plant species (Cermak et al. 2011; Christian
et al. 2010; Li et al. 2011; Mahfouz et al. 2011). Notably,
TALEN-mediated targeted mutagenesis (NHEJ-based) was
achieved in important crops (Clasen et al. 2016; Haun et al.
2014; Li et al. 2012; Liang et al. 2014; Lor et al. 2014;
Shan et al. 2015; Wang et al. 2014; Wendt et al. 2013).
Plant genes important for plant-pathogen interaction have
been knocked out to confer bacterial blight resistance in
rice and powdery mildew resistance in wheat (Li et al.
2012; Wang et al. 2014). Quality-related genes have been
selectively disrupted in soybean (Haun et al. 2014), maize
(Liang et al. 2014), rice (Ma et al. 2015a; Shan et al. 2015),
potato (Clasen et al. 2016; Sawai et al. 2014). In Nicotiana
benthamiana, a multiplex approach was pursued to inac-
tivate four genes involved in plant-specific protein glyco-
sylation, which allowed more desirable glycosylation
patterns for antibody production in plants (Li et al. 2016).
In the presence of appropriate homologous repair tem-
plates, TALEN-mediated gene targeting was shown in
tobacco protoplast-derived calli (Zhang et al. 2013), barley
leaf cells (Budhagatapalli et al. 2015), rice and tomato
plants (Cermak et al. 2015; Wang et al. 2015a). In rice,
single base gene editing of OsEPSPS was accomplished by
delivering a donor chimeric RNA/DNA oligonucleotide by
particle bombardment, while, in tomato, site-specific
insertion in front of ANT1 (a Myb transcription factor
involved in anthocyanin production) of a cassette con-
taining a selectable marker gene and the constitutive pro-
moter 35S was pursued by geminivirus-based delivery.
The fourth wave of genome editing approaches in plants
arrived in 2013 and was based on the application of engi-
neered clustered regularly interspaced short palindromic
repeats (CRISPR)/CRISPR-associated (Cas) systems. The
CRISPR/Cas system, used by bacteria and Archaea to
defend from invading viruses and plasmids, is based on the
formation of a tracrRNA–crRNA-Cas ribonucleoprotein
complex, in which specific RNAs recognize the invading
sequences and the Cas enzyme cleaves them. In 2012,
Jinek and colleagues demonstrated the possibility to com-
bine the functions of naturally separate tracrRNA and
crRNA in a single RNA molecule, dubbed ‘‘single guide
RNA (sgRNA),’’ an achievement which immediately
prompted biotechnological applications (Jinek et al. 2012).
Nine original papers in plants appeared in 2013, and around
60 in the following years (Fig. 5). Overall, about two-thirds
of the published studies are on important crop species,
which is indicative in utilizing CRISPR in plant breeding.
Indeed, CRISPR appears to be the most easily deployed
and efficient system among all the genome editing tools. A
large number of reviews have specifically dealt with basic
aspects and various features of CRISPR/Cas implementa-
tion in functional genomics and crop improvement
appeared in the last months [e.g. (Belhaj et al. 2015;
Bortesi and Fischer 2015; Chen and Gao 2015; Kumar and
Jain 2015; Quetier 2016; Raitskin and Patron 2015; Scha-
effer and Nakata 2015)]. Quite a number of studies were
carried out in the past few years in model Arabidopsis and
Nicotiana spp. as well as in crop species. Frequency of on-
and off-targeted modifications, mutation types, removal of
exogeneous sequences, sexual transmission of induced
somatic mutations were assessed with different approaches
(Feng et al. 2014; Gao et al. 2015; Li et al. 2013; Nekrasov
et al. 2013). Among crop species, rice is the leader for
deployment of CRISPR-based genome editing for potential
practical implications. The possibility to induce targeted
indels by NHEJ has been demonstrated in genes with
potential agronomic interest (e.g., tillering patterns, and
responses to pathogenic bacteria) selectively knocked-out
(Ikeda et al. 2016; Jiang et al. 2013; Miao et al. 2013; Shan
et al. 2013; Xu et al. 2014; Zhang et al. 2014; Zhou et al.
2014, 2015a). CRISPR/Cas was used to knockout various
genes with agronomic interest in wheat (resistance to
powdery mildew) or maize (phytate accumulation, male
fertility and herbicide resistance) (Liang et al. 2014; Shan
et al. 2013; Svitashev et al. 2015; Wang et al. 2014). The
general feasibility of the technology was investigated also
in sorghum and barley (Jiang et al. 2013; Lawrenson et al.
2015). In soybean, targeted mutagenesis experiments con-
sidered principally the possible implications for functional
genomics studies, also keeping in mind the ancient poly-
ploid nature of the species and the difficulty to knockout
multicopy genes (homeoalleles and members of gene
families). A. rhizogenes-mediated transformation inducing
hairy roots was used in most cases (Cai et al. 2015; Du
et al. 2016; Jacobs et al. 2015; Li et al. 2015; Sun et al.
2015). Among vegetables, proof-of-concept and functional
studies were carried out in tomato, potato and B. oleracea
(Brooks et al. 2014; Butler et al. 2015; Ito et al. 2015;
Lawrenson et al. 2015; Ron et al. 2014; Wang et al. 2015b).
The usefulness for tomato breeding of mutations with
1408 Plant Cell Rep (2016) 35:1401–1416
123
novel phenotype induced in RIN (encoding for a tran-
scription factor regulating fruit ripening) was discussed.
Finally, in woody species, CRISPR/Cas was used in sweet
orange (Jia and Wang 2014) and poplar (Fan et al. 2015;
Zhou et al. 2015b). In the latter species, two 4CL genes,
associated with lignin and flavonoid biosynthesis, were
targeted. Besides targeted mutagenesis due to NHEJ-based
repair of DSBs, CRISPR/Cas was soon employed also to
attempt HDR-dependent gene targeting. Based on the use
of ssDNA oligonucleotides or dsDNA plasmids as donor
repair molecules, first positive results were reported in
proof-of-concept studies in N. benthamiana (Li et al.
2013), rice (Shan et al. 2013) and Arabidopsis (Feng et al.
2014; Schiml et al. 2014). In the latter species, the in planta
strategy previously set up by the same group using the
I-SceI endonuclease (Fauser et al. 2012) was further
developed. More recently, HDR-mediated precise editing
of endogenous genes and/or insertion of exogeneous
sequences by CRISPR/Cas technology was demonstrated
also in maize, soybean and tomato, using particle bom-
bardment or geminivirus to deliver the CRISPR/Cas
expression system as well as the DNA repair templates. In
all cases, modified plants showed the expected phenotypes
(Cermak et al. 2015; Li et al. 2015; Svitashev et al. 2015).
The use of CRISPR/Cas for inducing virus resistance in
modified plants has been recently reported (Ali et al.
2015b; Baltes et al. 2015; Ji et al. 2015). Using both
transient and stable transformation in N. benthamiana and
A. thaliana, it was possible to direct sgRNA and Cas9
towards various geminivirus sequences, limiting their
infection titer in transfected plants. Although field trials are
necessary to establish plant resistance in natural environ-
ments and some concerns remain to be addressed (Cha-
parro-Garcia et al. 2015), these results further show the
versatility of CRISPR/Cas for biotechnological crop
improvement approaches. Clearly, RNA virus, the vast
majority of plant virus, are not readibily addressable with
such approach, although the recent development of novel
CRISPR/Cas able to recognize and cleave RNA molecules
is intriguing in that respect (O’Connell et al. 2014). Lately,
broad virus resistance has been shown in cucumber plants
in which the recessive eIF4E gene had been disrupted by
CRISPR/Cas (Chandrasekaran et al. 2016).
Application of CRISPR/Cas showed an incredible fast
development since its discovery. Nevertheless, the delivery
of the entire expression system, especially in case also a
donor repair molecule needs to be co-expressed, can be an
issue in plant cells. Several delivery methods have being
Fig. 5 The number of original research papers published between
1988 and 2015 reporting targeted genome modifications by technol-
ogy: HR homologous recombination-dependent gene targeting, REC
recombinase-mediated site-specific gene integration, ODM oligonu-
cleotide-directed mutagenesis, MN meganucleases, ZFN zinc finger
nucleases, TALEN transcription activator-like effector nucleases,
CRISPR Clustered Regularly Interspaced Short Palindromic
Repeats/CRISPR-associated systems. In parentheses, the total num-
ber of papers published for each technology
Plant Cell Rep (2016) 35:1401–1416 1409
123
tested, but response to in vitro culture systems (e.g. pro-
toplasts) can be limiting in some instances. The use of viral
vectors based on DNA (geminivirus) or RNA (TRV) was
advocated considering efficiency, transient expression,
possibility to skip difficult regeneration systems, multiplex
sgRNA expression, transmissibility of mutations to pro-
genies (Ali et al. 2015a; Baltes et al. 2014; Honig et al.
2015; Yin et al. 2015). The cargo ability of viral vectors,
however, must be considered. High targeted mutation
efficiency in combination with low off-target effects are
needed for crop breeding. Recent progress along these lines
include efficient bioinformatic tools to predict appropriate
targeting sites (Xie et al. 2014), optimized sgRNA structure
(Dang et al. 2015), novel Cas orthologues from bacteria
other than Streptococcus pyogenes or nucleases alternative
to Cas (e.g. Cpf1) with reduced size and improved speci-
ficity (Kleinstiver et al. 2016; Ran et al. 2015; Slaymaker
et al. 2016; Steinert et al. 2015; Zetsche et al. 2015),
parameters and elements for optimal expression of CRISPR
(Mao et al. 2016; Mikami et al. 2015a, b; Wang et al.
2015c; Yan et al. 2015), and procedures for increasing
multiplexing efficiency (Lowder et al. 2015; Ma et al.
2015b; Xie et al. 2015; Xing et al. 2014). Finally, the direct
delivery of ribonucloproteins, important also for regulatory
reasons, has been recently achieved in various plant species
(Woo et al. 2015).
Conclusions and perspectives
Since the rediscovery of Mendel’s laws at the beginning of
the 20th century, plant breeding has incorporated and
profited from new scientific and technological innovations
in genetics and biology, resulting in increased efficiency.
Overall, it is estimated that genetic improvement has
contributed by about 50 % to the increase of crop plant
productivity achieved so far (Xu 2010). For about one
century, however, the selection of superior genotypes was
based on phenotypic selection and inference of genotypic
value through the assessment of phenotypic value. Only at
the end of last century have the development of molecular
markers and genetic engineering techniques contributed to
a more direct relationship between genotype and pheno-
type, with improved selection and prediction efficiency.
The first generation of genetically modified plants were,
however, based on the transfer and random insertion of a
single transgene and a marker gene into the host plant
genomes, with possible negative consequences on the
ability to have constant and predictable results in terms of
gene expression and phenotypic performance. Multigenic
traits were not addressed. In this century, increased
knowledge about the genome structure and function has
laid the foundation not only for more targeted and efficient
methods to select parental and recombinant genotypes in
crosses, but also for the development of so-called second-
generation biotechnologies and their application to
breeding.
Technologies for targeted mutagenesis and gene inser-
tion in higher plants have been a highly desirable objective
for almost 30 years, and we are experiencing rapid pro-
gress (Fig. 5). Homologous recombination-dependent gene
targeting, however, has only been accomplished in rice and
in a limited number of laboratories, where it could be
successfully used to precisely modify several traits of
interest. Its efficiency is too low for general applicability.
Similarly, the use of oligonucleotides to precisely direct
mutagenesis in preselected sequences has produced inter-
esting results in some cases, but it has not been widely
deployed in crops. Heterologous recombinases appear to be
powerful tools for post-transformation removal of marker
genes as well as other unwanted sequences. On the other
hand, techniques for recombinase-mediated site-specific
gene integration are likely limited. Despite the interesting
results achieved so far, the prerequisite of first randomly
introducing target recombination sites in the host genome
and then select ‘‘better’’ plants for subsequent transfor-
mation is clumsy. We know of only one example where
recombinases were used for gene integration in crops for
agronomically relevant traits (Li et al. 2010). Also native
homing endonucleases have the inherent limitation of the
previous insertion of the recognition site. Hence, the pos-
sibility to engineer chimeric designer nucleases able to
target virtually any genomic site, and use them for inducing
DSBs, opened a new scenario both for applied breeding
and functional studies. Indeed, the repair of induced DSBs
could be harnessed for different genome editing approa-
ches, including various forms of targeted mutagenesis as
well as efficient gene replacement/stacking. Out of 212
studies published between 1988 and 2015, almost two-
thirds were based on nuclease induced-DSBs. Thus, it
appears these techniques are quite powerful. Besides the
vast number of applications reported in the literature and
described above, genome editing approaches in combina-
tion with other new plant breeding techniques (e.g. cisge-
nesis), could be used also for the exploitation of plant
genetic resources in crop improvement (Cardi 2016).
Albeit it is the most recent among the new technologies
available for precise editing of plant genomes, CRISPR has
already been extensively used in molecular plant breeding.
The large majority of plant genome editing studies have
occurred in the past 3 years. CRISPR and commensurate
increase in genomics information in crops should enable
rapid genetic improvement over the next 20 years. Now we
must examine the bottlenecks for both research and agri-
cultural application of these tools. Certainly, some topics
need further study and will soon be resolved. These include
1410 Plant Cell Rep (2016) 35:1401–1416
123
locus targeting, transmissibility of induced mutations, fre-
quency of off-target effects, and the public acceptability of
gene drives. The most likely scientific bottleneck we
foresee is the current inherent limitations in crop trans-
formation technologies. Topping that might be the socio-
political landscape about GMOs and regulatory issues.
Commercial development and implementation in plant
breeding of targeted genome modification approaches may
ultimately depend on the whims of the public and the
politicians who serve them (Huang et al. 2016; Lusser et al.
2012).
Author contribution statement Both authors wrote and
reviewed the manuscript.
Acknowledgments The support of the ‘‘GenHort’’ project (‘‘Ad-
ding value to elite Campania horticultural crops by advanced genomic
technologies,’’ the Italian Ministry of Research and University-MIUR
PON02_00395_3215002) to TC is acknowledged. CNS thanks
funding from a USDA HATCH grant, a USDA NIFA Biotechnology
Risk Assessment grant, and funding from the Ivan Racheff endow-
ment at the University of Tennessee. TC dedicates this work to Prof.
Luigi Monti, University of Naples ‘‘Federico II,’’ on occasion of his
80th birthday.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts
of interest.
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