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

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(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

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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

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‘‘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

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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

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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.

References

Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Zeitler B,

Amora R, Corbin DR, Miles RR, Arnold NL, Strange TL,

Simpson MA, Cao Z, Carroll C, Pawelczak KS, Blue R, West K,

Rowland LM, Perkins D, Samuel P, Dewes CM, Shen L, Sriram

S, Evans SL, Rebar EJ, Zhang L, Gregory PD, Urnov FD, Webb

SR, Petolino JF (2013) Trait stacking via targeted genome

editing. Plant Biotechnol J 11:1126–1134

Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of

DNA into wild-type and mutant lox sites placed in the plant

genome. Plant J 7:649–659

Ali Z, Abul-faraj A, Li L, Ghosh N, Piatek M, Mahjoub A, Aouida M,

Piatek A, Baltes NJ, Voytas DF, Dinesh-Kumar S, Mahfouz MM

(2015a) Efficient virus-mediated genome editing in plants using

the CRISPR/Cas9 system. Mol Plant 8:1288–1291

Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM

(2015b) CRISPR/Cas9-mediated viral interference in plants.

Genome Biol 16:238

Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF (2014)

DNA replicons for plant genome engineering. Plant Cell

26:151–163

Baltes NJ, Hummel AW, Konecna E, Cegan R, Bruns AN, Bisaro

DM, Voytas DF (2015) Conferring resistance to geminiviruses

with the CRISPR–Cas prokaryotic immune system. Nature

Plants 1:15145

Beetham PR, Kipp PB, Sawycky XL, Arntzen CJ, May GD (1999) A

tool for functional plant genomics: chimeric RNA/DNA

oligonucleotides cause in vivo gene-specific mutations. Proc

Natl Acad Sci USA 96:8774–8778

Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V

(2015) Editing plant genomes with CRISPR/Cas9. Curr Opin

Biotechnol 32:76–84

Bevan MW, Flavell RB, Chilton M-D (1983) A chimaeric antibiotic

resistance gene as a selectable marker for plant cell transforma-

tion. Nature 304:184–187

Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant

genome editing and beyond. Biotechnol Adv 33:41–52

Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene

editing in tomato in the first generation using the clustered

regularly interspaced short palindromic repeats/CRISPR-associ-

ated9 system. Plant Physiol 166:1292–1297

Budhagatapalli N, Rutten T, Gurushidze M, Kumlehn J, Hensel G

(2015) Targeted modification of gene function exploiting

homology-directed repair of TALEN-mediated double-strand

breaks in barley. G3 (Bethesda) 5:1857–1863

Butaye KMJ, Cammue BPA, Delaure SL, De Bolle MFC (2005)

Approaches to minimize variation of transgene expression in

plants. Mol Breed 16:79–91

Butler NM, Atkins PA, Voytas DF, Douches DS (2015) Generation and

inheritance of targeted mutations in potato (Solanum tuberosum

L.) using the CRISPR/Cas system. PLoS One 10:e0144591

Cai Y, Chen L, Liu X, Sun S, Wu C, Jiang B, Han T, Hou W (2015)

CRISPR/Cas9-mediated genome editing in soybean hairy roots.

PLoS One 10:e0136064

Cardi T (2016) Cisgenesis and genome editing: combining concepts

and efforts for a smarter use of genetic resources in crop

breeding. Plant Breed. doi:10.1111/pbr.12345

Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C,

Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011)

Efficient design and assembly of custom TALEN and other

TAL effector-based constructs for DNA targeting. Nucleic Acids

Res 39:e82

Cermak T, Baltes NJ, Cegan R, Zhang Y, Voytas DF (2015) High-

frequency, precise modification of the tomato genome. Genome

Biol 16:232

Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C,

Pearlsman M, Sherman A, Arazi T, Gal-On A (2016) Develop-

ment of broad virus resistance in non-transgenic cucumber using

CRISPR/Cas9 technology. Mol Plant Pathol

Chaparro-Garcia A, Kamoun S, Nekrasov V (2015) Boosting plant

immunity with CRISPR/Cas. Genome Biol 16:254

Chawla R, Ariza-Nieto M, Wilson AJ, Moore SK, Srivastava V

(2006) Transgene expression produced by biolistic-mediated,

site-specific gene integration is consistently inherited by the

subsequent generations. Plant Biotechnol J 4:209–218

Chen K, Gao C (2014) Targeted genome modification technologies

and their applications in crop improvements. Plant Cell Rep

33:575–583

Chen K, Gao C (2015) Developing CRISPR technology in major crop

plants. In: Zhang F, Puchta H, Thomson JG (eds) Advances in

new technology for targeted modification of plant genomes.

Springer, New York, pp 145–159

Chilton MD, Que Q (2003) Targeted integration of T-DNA into the

tobacco genome at double-stranded breaks: new insights on the

mechanism of T-DNA integration. Plant Physiol 133:956–965

Choi S, Begum D, Koshinsky H, Ow DW, Wing RA (2000) A new

approach for the identification and cloning of genes: the

pBACwich system using Cre/lox site-specific recombination.

Nucleic Acids Res 28:E19

Christian M, Voytas DF (2015) Engineered TAL effector proteins:

Versatile reagents for manipulating plant genomes. In: Zhang F,

Puchta H, Thomson JG (eds) Advances in new technology for

targeted modification of plant genomes. Springer, New York,

pp 55–72

Plant Cell Rep (2016) 35:1401–1416 1411

123

Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A,

Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand

breaks with TAL effector nucleases. Genetics 186:757–761

Clasen BM, Stoddard TJ, Luo S, Demorest ZL, Li J, Cedrone F,

Tibebu R, Davison S, Ray EE, Daulhac A, Coffman A,

Yabandith A, Retterath A, Haun W, Baltes NJ, Mathis L,

Voytas DF, Zhang F (2016) Improving cold storage and

processing traits in potato through targeted gene knockout. Plant

Biotechnol J 14:169–176

Curtin SJ, Zhang F, Sander JD, Haun WJ, Starker C, Baltes NJ, Reyon

D, Dahlborg EJ, Goodwin MJ, Coffman AP, Dobbs D, Joung JK,

Voytas DF, Stupar RM (2011) Targeted mutagenesis of dupli-

cated genes in soybean with zinc-finger nucleases. Plant Physiol

156:466–473

Da Ines O, White CI (2013) Gene site-specific insertion in plants. In:

Renault S, Duchateau P (eds) Topics in current genetics.

Springer, Netherlands, pp 287–315

Daboussi F, Stoddard TJ, Zhang F (2015) Engineering meganuclease

for precise plant genome modification. In: Zhang F, Puchta H,

Thomson JG (eds) Advances in new technology for targeted

modification of plant genomes. Springer, New York, pp 21–38

Dale EC, Ow DW (1990) Intra- and intramolecular site-specific

recombination in plant cells mediated by bacteriophage P1

recombinase. Gene 91:79–85

Dale EC, Ow DW (1991) Gene transfer with subsequent removal of

the selection gene from the host genome. Proc Natl Acad Sci

USA 88:10558–10562

Dang TT, Shimatani Z, Kawano Y, Terada R, Shimamoto K (2013)

Gene editing a constitutively active OsRac1 by homologous

recombination-based gene targeting induces immune responses

in rice. Plant Cell Physiol 54:2058–2070

Dang Y, Jia G, Choi J, Ma H, Anaya E, Ye C, Shankar P, Wu H

(2015) Optimizing sgRNA structure to improve CRISPR-Cas9

knockout efficiency. Genome Biol 16:280

Day CD (2000) Transgene integration into the same chromosome

location can produce alleles that express at a predictable level, or

alleles that are differentially silenced. Genes Dev 14:2869–2880

De Paepe A, De Buck S, Nolf J, Van Lerberge E, Depicker A (2013)

Site-specific T-DNA integration in Arabidopsis thaliana medi-

ated by the combined action of CRE recombinase and uC31integrase. Plant J 75:172–184

de Pater S, Neuteboom LW, Pinas JE, Hooykaas PJ, van der Zaal BJ

(2009) ZFN-induced mutagenesis and gene-targeting in Ara-

bidopsis through Agrobacterium-mediated floral dip transforma-

tion. Plant Biotechnol J 7:821–835

de Pater S, Pinas JE, Hooykaas PJ, van der Zaal BJ (2013) ZFN-

mediated gene targeting of the Arabidopsis protoporphyrinogen

oxidase gene through Agrobacterium-mediated floral dip trans-

formation. Plant Biotechnol J 11:510–515

D’Halluin K, Vanderstraeten C, Stals E, Cornelissen M, Ruiter R

(2008) Homologous recombination: a basis for targeted genome

optimization in crop species such as maize. Plant Biotechnol J

6:93–102

D’Halluin K, Vanderstraeten C, Van Hulle J, Rosolowska J, Van Den

Brande I, Pennewaert A, D’Hont K, Bossut M, Jantz D, Ruiter R,

Broadhvest J (2013) Targeted molecular trait stacking in cotton

through targeted double-strand break induction. Plant Biotechnol

J 11:933–941

Djukanovic V, Smith J, Lowe K, Yang M, Gao H, Jones S, Nicholson

MG, West A, Lape J, Bidney D, Carl Falco S, Jantz D,

Alexander Lyznik L (2013) Male-sterile maize plants produced

by targeted mutagenesis of the cytochrome P450-like gene

(MS26) using a re-designed I-CreI homing endonuclease. Plant J

76:888–899

Dong C, Beetham P, Vincent K, Sharp P (2006) Oligonucleotide-

directed gene repair in wheat using a transient plasmid gene

repair assay system. Plant Cell Rep 25:457–465

Du H, Zeng X, Zhao M, Cui X, Wang Q, Yang H, Cheng H, Yu D

(2016) Efficient targeted mutagenesis in soybean by TALENs

and CRISPR/Cas9. J Biotechnol 217:90–97

Ebinuma H, Nakahama K, Nanto K (2015) Enrichments of gene

replacement events by Agrobacterium-mediated recombinase-

mediated cassette exchange. Mol Breed 35:82

Endo M, Osakabe K, Ono K, Handa H, Shimizu T, Toki S (2007)

Molecular breeding of a novel herbicide-tolerant rice by gene

targeting. Plant J 52:157–166

Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, Luo K (2015) Efficient

CRISPR/Cas9-mediated targeted mutagenesis in Populus in the

first generation. Sci Rep 5:12217

Fauser F, Roth N, Pacher M, Ilg G, Sanchez-Fernandez R, Biesgen C,

Puchta H (2012) In planta gene targeting. Proc Natl Acad Sci

USA 109:7535–7540

Feldmann KA (1999) The greening of chimeric oligonucleotides. Nat

Biotechnol 17:857–858

Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang DL, Wang Z, Zhang Z,

Zheng R, Yang L, Zeng L, Liu X, Zhu JK (2014) Multigener-

ation analysis reveals the inheritance, specificity, and patterns of

CRISPR/Cas-induced gene modifications in Arabidopsis. Proc

Natl Acad Sci USA 111:4632–4637

Fichtner F, Urrea Castellanos R, Ulker B (2014) Precision genetic

modifications: a new era in molecular biology and crop

improvement. Planta 239:921–939

Fladung M, Becker D (2010) Targeted integration and removal of

transgenes in hybrid aspen (Populus tremula L. x P. tremuloides

Michx.) using site-specific recombination systems. Plant Biol

(Stuttg) 12:334–340

Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP,

Bittner ML, Brand LA, Fink CL, Fry JS, Galluppi GR, Goldberg

SB, Hoffmann NL, Woo SC (1983) Expression of bacterial

genes in plant cells. Proc Natl Acad Sci USA 80:4803–4807

Gallego ME, Sirand-Pugnet P, White CI (1999) Positive–negative

selection and T-DNA stability in Arabidopsis transformation.

Plant Mol Biol 39:83–93

Gao H, Smith J, Yang M, Jones S, Djukanovic V, Nicholson MG,

West A, Bidney D, Falco SC, Jantz D, Lyznik LA (2010)

Heritable targeted mutagenesis in maize using a designed

endonuclease. Plant J 61:176–187

Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, Wu Y, Zhao P, Xia Q

(2015) CRISPR/Cas9-mediated targeted mutagenesis in Nico-

tiana tabacum. Plant Mol Biol 87:99–110

Gocal GFW, Schopke C, Beetham PR (2015) Oligo-mediated targeted

gene editing. In: Zhang F, Puchta H, Thomson JG (eds)

Advances in new technology for targeted modification of plant

genomes. Springer, New York, pp 73–89

Hanin M, Volrath S, Bogucki A, Briker M, Ward E, Paszkowski J

(2001) Gene targeting in Arabidopsis. Plant J 28:671–677

Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E,

Retterath A, Stoddard T, Juillerat A, Cedrone F, Mathis L,

Voytas DF, Zhang F (2014) Improved soybean oil quality by

targeted mutagenesis of the fatty acid desaturase 2 gene family.

Plant Biotechnol J 12:934–940

Herrera-Estrella L, Depicker A, Van Montagu M, Schell J (1983)

Expression of chimaeric genes transferred into plant cells using a

Ti-plasmid-derived vector. Nature 303:209–213

Honig A, Marton I, Rosenthal M, Smith JJ, Nicholson MG, Jantz D,

Zuker A, Vainstein A (2015) Transient expression of virally

delivered meganuclease in planta generates inherited genomic

deletions. Mol Plant 8:1292–1294

1412 Plant Cell Rep (2016) 35:1401–1416

123

Hou L, Yau YY, Wei J, Han Z, Dong Z, Ow DW (2014) An open-

source system for in planta gene stacking by Bxb1 and Cre

recombinases. Mol Plant 7:1756–1765

Huang S, Weigel D, Beachy RN, Li J (2016) A proposed regulatory

framework for genome-edited crops. Nat Genet 48:109–111

Iida S, Terada R (2005) Modification of endogenous natural genes by

gene targeting in rice and other higher plants. Plant Mol Biol

59:205–219

Ikeda T, Tanaka W, Mikami M, Endo M, Hirano HY (2016)

Generation of artificial drooping leaf mutants by CRISPR-Cas9

technology in rice. Genes Genet Syst 90:231–235

Ito Y, Nishizawa-Yokoi A, Endo M, Mikami M, Toki S (2015)

CRISPR/Cas9-mediated mutagenesis of the RIN locus that

regulates tomato fruit ripening. Biochem Biophys Res Commun

467:76–82

Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA (2015) Targeted

genome modifications in soybean with CRISPR/Cas9. BMC

Biotechnol 15:16

Ji X, Zhang H, Zhang Y, Wang Y, Gao C (2015) Establishing a

CRISPR–Cas-like immune system conferring DNA virus resis-

tance in plants. Nature Plants 1:15144

Jia H, Wang N (2014) Targeted genome editing of sweet orange using

Cas9/sgRNA. PLoS One 9:e93806

Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013)

Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene

modification in Arabidopsis, tobacco, sorghum and rice. Nucleic

Acids Res 41:e188

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E

(2012) A programmable dual-RNA-guided DNA endonuclease

in adaptive bacterial immunity. Science 337:816–821

Kempin SA, Liljegren SJ, Block LM, Rounsley SD, Yanofsky MF,

Lam E (1997) Targeted disruption in Arabidopsis. Nature

389:802–803

Kerbach S, Lorz H, Becker D (2005) Site-specific recombination in

Zea mays. Theor Appl Genet 111:1608–1616

Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng

Z, Joung JK (2016) High-fidelity CRISPR-Cas9 nucleases with

no detectable genome-wide off-target effects. Nature

529:490–495

Koch A, Kogel KH (2014) New wind in the sails: improving the

agronomic value of crop plants through RNAi-mediated gene

silencing. Plant Biotechnol J 12:821–831

Kochevenko A, Willmitzer L (2003) Chimeric RNA/DNA oligonu-

cleotide-based site-specific modification of the tobacco aceto-

lactate syntase gene. Plant Physiol 132:174–184

Kumar V, Jain M (2015) The CRISPR-Cas system for plant genome

editing: advances and opportunities. J Exp Bot 66:47–57

Kumar S, AlAbed D, Worden A, Novak S, Wu H, Ausmus C, Beck

M, Robinson H, Minnicks T, Hemingway D, Lee R, Skaggs N,

Wang L, Marri P, Gupta M (2015) A modular gene targeting

system for sequential transgene stacking in plants. J Biotechnol

207:12–20

Lawrenson T, Shorinola O, Stacey N, Li C, Ostergaard L, Patron N,

Uauy C, Harwood W (2015) Induction of targeted, heritable mu-

tations in barley and Brassica oleracea using RNA-guided Cas9

nuclease. Genome Biol 16:258

Lee J, Chung JH, Kim HM, Kim DW, Kim H (2016) Designed

nucleases for targeted genome editing. Plant Biotechnol J

14:448–462

Li Z, Xing A, Moon BP, McCardell RP, Mills K, Falco SC (2009)

Site-specific integration of transgenes in soybean via recombi-

nase-mediated DNA cassette exchange. Plant Physiol

151:1087–1095

Li Z, Moon BP, Xing A, Liu ZB, McCardell RP, Damude HG, Falco

SC (2010) Stacking multiple transgenes at a selected genomic

site via repeated recombinase-mediated DNA cassette

exchanges. Plant Physiol 154:622–631

Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang

B (2011) TAL nucleases (TALNs): hybrid proteins composed of

TAL effectors and FokI DNA-cleavage domain. Nucleic Acids

Res 39:359–372

Li T, Liu B, Spalding MH, Weeks DP, Yang B (2012) High-efficiency

TALEN-based gene editing produces disease-resistant rice. Nat

Biotechnol 30:390–392

Li J-F, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church

GM, Sheen J (2013) Multiplex and homologous recombination-

mediated genome editing in Arabidopsis and Nicotiana ben-

thamiana using guide RNA and Cas9. Nat Biotechnol

31:688–691

Li Z, Liu ZB, Xing A, Moon BP, Koellhoffer JP, Huang L, Ward RT,

Clifton E, Falco SC, Cigan AM (2015) Cas9-guide RNA directed

genome editing in soybean. Plant Physiol 169:960–970

Li J, Stoddard TJ, Demorest ZL, Lavoie PO, Luo S, Clasen BM,

Cedrone F, Ray EE, Coffman AP, Daulhac A, Yabandith A,

Retterath AJ, Mathis L, Voytas DF, D’Aoust MA, Zhang F

(2016) Multiplexed, targeted gene editing in Nicotiana ben-

thamiana for glyco-engineering and monoclonal antibody pro-

duction. Plant Biotechnol J 14:533–542

Liang Z, Zhang K, Chen K, Gao C (2014) Targeted mutagenesis in

Zea mays using TALENs and the CRISPR/Cas system. J Genet

Genom 41:63–68

Lloyd A, Plaisier CL, Carroll D, Drews GN (2005) Targeted

mutagenesis using zinc-finger nucleases in Arabidopsis. Proc

Natl Acad Sci USA 102:2232–2237

Lor VS, Starker CG, Voytas DF, Weiss D, Olszewski NE (2014)

Targeted mutagenesis of the tomato PROCERA gene using

transcription activator-like effector nucleases. Plant Physiol

166:1288–1291

Louwerse JD, van Lier MC, van der Steen DM, de Vlaam CM,

Hooykaas PJ, Vergunst AC (2007) Stable recombinase-mediated

cassette exchange in Arabidopsis using Agrobacterium tumefa-

ciens. Plant Physiol 145:1282–1293

Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X, Zheng X,

Voytas DF, Hsieh TF, Zhang Y, Qi Y (2015) A CRISPR/Cas9

toolbox for multiplexed plant genome editing and transcriptional

regulation. Plant Physiol 169:971–985

Lusser M, Parisi C, Plan D, Rodriguez-Cerezo E (2012) Deployment

of new biotechnologies in plant breeding. Nat Biotechnol

30:231–239

Lutz KA, Corneille S, Azhagiri AK, Svab Z, Maliga P (2004) A novel

approach to plastid transformation utilizes the phiC31 phage

integrase. Plant J 37:906–913

Lyznik LA, Gordon-Kamm WJ, Tao Y (2003) Site-specific recom-

bination for genetic engineering in plants. Plant Cell Rep

21:925–932

Ma L, Zhu F, Li Z, Zhang J, Li X, Dong J, Wang T (2015a) TALEN-

based mutagenesis of lipoxygenase LOX3 enhances the storage

tolerance of rice (Oryza sativa) seeds. PLoS One 10:e0143877

Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li

H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen

L, Zhao X, Dong Z, Liu Y-G (2015b) A robust CRISPR/Cas9

system for convenient, high-efficiency multiplex genome editing

in monocot and dicot plants. Mol Plant 8:1274–1284

Mahfouz MM, Li L (2011) TALE nucleases and next generation GM

crops. GM Crops 2:99–103

Mahfouz MM, Li L, Shamimuzzaman M, Wibowo A, Fang X, Zhu

JK (2011) De novo-engineered transcription activator-like

effector (TALE) hybrid nuclease with novel DNA binding

specificity creates double-strand breaks. Proc Natl Acad Sci

USA 108:2623–2628

Plant Cell Rep (2016) 35:1401–1416 1413

123

Mao Y, Zhang Z, Feng Z, Wei P, Zhang H, Botella JR, Zhu JK (2016)

Development of germ-line-specific CRISPR-Cas9 systems to

improve the production of heritable gene modifications in

Arabidopsis. Plant Biotechnol J 14:519–532

Marton I, Zuker A, Shklarman E, Zeevi V, Tovkach A, Roffe S,

Ovadis M, Tzfira T, Vainstein A (2010) Nontransgenic genome

modification in plant cells. Plant Physiol 154:1079–1087

Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu

L-J (2013) Targeted mutagenesis in rice using CRISPR-Cas

system. Cell Res 23:1233–1236

Michael TP, VanBuren R (2015) Progress, challenges and the future

of crop genomes. Curr Opin Plant Biol 24:71–81

Mikami M, Toki S, Endo M (2015a) Comparison of CRISPR/Cas9

expression constructs for efficient targeted mutagenesis in rice.

Plant Mol Biol 88:561–572

Mikami M, Toki S, Endo M (2015b) Parameters affecting frequency

of CRISPR/Cas9 mediated targeted mutagenesis in rice. Plant

Cell Rep 34:1807–1815

Moritoh S, Eun CH, Ono A, Asao H, Okano Y, Yamaguchi K,

Shimatani Z, Koizumi A, Terada R (2012) Targeted disruption

of an orthologue of DOMAINS REARRANGED METHYLASE

2, OsDRM2, impairs the growth of rice plants by abnormal DNA

methylation. Plant J 71:85–98

Murai N, Kemp JD, Sutton DW, Murray MG, Slightom JL, Merlo DJ,

Reichert NA, Sengupta-Gopalan C, Stock CA, Barker RF, Hall

TC (1983) Phaseolin gene from bean is expressed after transfer

to sunflower via tumor-inducing plasmid vectors. Science

222:476–482

Nandy S, Srivastava V (2011) Site-specific gene integration in rice

genome mediated by the FLP-FRT recombination system. Plant

Biotechnol J 9:713–721

Nandy S, Srivastava V (2012) Marker-free site-specific gene

integration in rice based on the use of two recombination

systems. Plant Biotechnol J 10:904–912

Nandy S, Zhao S, Pathak BP, Manoharan M, Srivastava V (2015)

Gene stacking in plant cell using recombinases for gene

integration and nucleases for marker gene deletion. BMC

Biotechnol 15:93

Nanto K, Ebinuma H (2008) Marker-free site-specific integration

plants. Transgenic Res 17:337–344

Nanto K, Yamada-Watanabe K, Ebinuma H (2005) Agrobacterium-

mediated RMCE approach for gene replacement. Plant Biotech-

nol J 3:203–214

Nanto K, Sato K, Katayama Y, Ebinuma H (2009) Expression of a

transgene exchanged by the recombinase-mediated cassette

exchange (RMCE) method in plants. Plant Cell Rep 28:777–785

Nekrasov V, Staskawicz B, Weigel D, Jones JDG, Kamoun S (2013)

Targeted mutagenesis in the model plant Nicotiana benthamiana

using Cas9 RNA-guided endonuclease. Nat Biotechnol

31:691–693

Nishizawa-Yokoi A, Endo M, Ohtsuki N, Saika H, Toki S (2015a)

Precision genome editing in plants via gene targeting and

piggyBac-mediated marker excision. Plant J 81:160–168

Nishizawa-Yokoi A, Nonaka S, Osakabe K, Saika H, Toki S (2015b)

A universal positive-negative selection system for gene targeting

in plants combining an antibiotic resistance gene and its

antisense RNA. Plant Physiol 169:362–370

O’Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M,

Doudna JA (2014) Programmable RNA recognition and cleav-

age by CRISPR/Cas9. Nature 516:263–266

Odell J, Caimi P, Sauer B, Russell S (1990) Site-directed recombi-

nation in the genome of transgenic tobacco. Mol Gen Genet

223:369–378

Okuzaki A, Toriyama K (2004) Chimeric RNA/DNA oligonu-

cleotide-directed gene targeting in rice. Plant Cell Rep

22:509–512

Ono A, Yamaguchi K, Fukada-Tanaka S, Terada R, Mitsui T, Iida S

(2012) A null mutation of ROS1a for DNA demethylation in rice

is not transmittable to progeny. Plant J 71:564–574

Osakabe Y, Osakabe K (2015) Genome editing with engineered

nucleases in plants. Plant Cell Physiol 56:389–400

Osakabe K, Osakabe Y, Toki S (2010) Site-directed mutagenesis in

Arabidopsis using custom-designed zinc finger nucleases. Proc

Natl Acad Sci USA 107:12034–12039

Osakabe K, Nishizawa-Yokoi A, Ohtsuki N, Osakabe Y, Toki S

(2014) A mutated cytosine deaminase gene, codA (D314A), as

an efficient negative selection marker for gene targeting in rice.

Plant Cell Physiol 55:658–665

Ow DW (2002) Recombinase-directed plant transformation for the

post-genomic era. Plant Mol Biol 48:183–200

Ow DW (2011) Recombinase-mediated gene stacking as a transfor-

mation operating system. J Integr Plant Biol 53:512–519

Ow DW (2016) The long road to recombinase-mediated plant

transformation. Plant Biotechnol J 14:441–447

Ozawa K, Wakasa Y, Ogo Y, Matsuo K, Kawahigashi H, Takaiwa F

(2012) Development of an efficient agrobacterium-mediated

gene targeting system for rice and analysis of rice knockouts

lacking granule-bound starch synthase (Waxy) and beta1,2-

xylosyltransferase. Plant Cell Physiol 53:755–761

Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting

in plants. EMBO J 7:4021–4026

Peer R, Rivlin G, Golobovitch S, Lapidot M, Gal-On A, Vainstein A,

Tzfira T, Flaishman MA (2015) Targeted mutagenesis using

zinc-finger nucleases in perennial fruit trees. Planta 241:941–951

Petolino JF (2015) Genome editing in plants via designed zinc finger

nucleases. In Vitro Cell Dev Biol-Plant 51:1–8

Puchta H, Fauser F (2013) Gene targeting in plants: 25 years later. Int

J Dev Biol 57:629–637

Puchta H, Fauser F (2014) Synthetic nucleases for genome engineer-

ing in plants: prospects for a bright future. Plant J 78:727–741

Puchta H, Fauser F (2015) Double-strand break repair and its

application to genome engineering in plants. In: Zhang F, Puchta

H, Thomson JG (eds) Advances in new technology for targeted

modification of plant genomes. Springer, New York, pp 1–20

Puchta H, Dujon B, Hohn B (1993) Homologous recombination in

plant-cells is enhanced by in vivo induction of double-strand

breaks into DNA by a site-specific endonuclease. Nucleic Acids

Res 21:5034–5040

Puchta H, Dujon B, Hohn B (1996) Two different but related

mechanisms are used in plants for the repair of genomic double-

strand breaks by homologous recombination. Proc Natl Acad Sci

USA 93:5055–5060

Qi Y (2015) High efficient genome modification by designed zinc

finger nuclease. In: Zhang F, Puchta H, Thomson JG (eds)

Advances in new technology for targeted modification of plant

genomes. Springer, New York, pp 39–53

Quetier F (2016) The CRISPR-Cas9 technology: Closer to the

ultimate toolkit for targeted genome editing. Plant Sci 242:65–76

Raitskin O, Patron NJ (2015) Multi-gene engineering in plants with

RNA-guided Cas9 nuclease. Curr Opin Biotechnol 37:69–75

Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ,

Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp

PA, Zhang F (2015) In vivo genome editing using Staphylococ-

cus aureus Cas9. Nature 520:186–191

Rinaldo AR, Ayliffe M (2015) Gene targeting and editing in crop

plants: a new era of precision opportunities. Mol Breed 35:1–15

Rivera-Torres N, Kmiec EB (2016) Genetic spell-checking: gene

editing using single-stranded DNA oligonucleotides. Plant

Biotechnol J 14:463–470

Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K,

Garcha J, Winte S, Masson H, Inagaki S, Federici F, Sinha N,

Deal RB, Bailey-Serres J, Brady SM (2014) Hairy root

1414 Plant Cell Rep (2016) 35:1401–1416

123

transformation using Agrobacterium rhizogenes as a tool for

exploring cell type-specific gene expression and function using

tomato as a model. Plant Physiol 166:455–469

Ruiter R, van den Brande I, Stals E, Delaure S, Cornelissen M,

D’Halluin K (2003) Spontaneous mutation frequency in plants

obscures the effect of chimeraplasty. Plant Mol Biol 53:675–689

Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a

transgene from the plant genome. Mol Gen Genet 234:49–59

Saika H, Oikawa A, Matsuda F, Onodera H, Saito K, Toki S (2011)

Application of gene targeting to designed mutation breeding of

high-tryptophan rice. Plant Physiol 156:1269–1277

Salomon S, Puchta H (1998) Capture of genomic and T-DNA

sequences during double-strand break repair in somatic plant

cells. EMBO J 17:6086–6095

Sauer B (1987) Functional expression of the cre-lox site-specific

recombination system in the yeast Saccharomyces cerevisiae.

Mol Cell Biol 7:2087–2096

Sauer B, Henderson N (1988) Site-specific DNA recombination in

mammalian-cells by the Cre recombinase of bacteriophage-p1.

Proc Natl Acad Sci USA 85:5166–5170

Sauer NJ, Mozoruk J, Miller RB, Warburg ZJ, Walker KA, Beetham

PR, Schopke CR, Gocal GF (2016) Oligonucleotide-directed

mutagenesis for precision gene editing. Plant Biotechnol J

14:496–502

Sawai S, Ohyama K, Yasumoto S, Seki H, Sakuma T, Yamamoto T,

Takebayashi Y, Kojima M, Sakakibara H, Aoki T, Muranaka T,

Saito K, Umemoto N (2014) Sterol side chain reductase 2 is a

key enzyme in the biosynthesis of cholesterol, the common

precursor of toxic steroidal glycoalkaloids in potato. Plant Cell

26:3763–3774

Schaeffer SM, Nakata PA (2015) CRISPR/Cas9-mediated genome

editing and gene replacement in plants: transitioning from lab to

field. Plant Sci 240:130–142

Schiml S, Fauser F, Puchta H (2014) The CRISPR/Cas system can be

used as nuclease for in planta gene targeting and as paired

nickases for directed mutagenesis in Arabidopsis resulting in

heritable progeny. Plant J 80:1139–1150

Schneider K, Schiermeyer A, Dolls A, Koch N, Herwartz D, Kirchhoff

J, Fischer R, Russell SM, Cao Z, Corbin DR, Sastry-Dent L,

Ainley WM, Webb SR, Schinkel H, Schillberg S (2016) Targeted

gene exchange in plant cells mediated by a zinc finger nuclease

double cut. Plant Biotechnol J 14:1151–1160

Schouten HJ, Krens FA, Jacobsen E (2006) Cisgenic plants are

similar to traditionally bred plants: international regulations for

genetically modified organisms should be altered to exempt

cisgenesis. EMBO Rep 7:750–753

Shaked H, Melamed-Bessudo C, Levy AA (2005) High-frequency

gene targeting in Arabidopsis plants expressing the yeast RAD54

gene. Proc Natl Acad Sci USA 102:12265–12269

Shan QW, Wang YP, Li J, Zhang Y, Chen KL, Liang Z, Zhang K, Liu

JX, Xi JJ, Qiu JL, Gao CX (2013) Targeted genome modification

of crop plants using a CRISPR-Cas system. Nat Biotechnol

31:686–688

Shan Q, Zhang Y, Chen K, Zhang K, Gao C (2015) Creation of

fragrant rice by targeted knockout of the OsBADH2 gene using

TALEN technology. Plant Biotechnol J 13:791–800

Sheehy RE, Kramer M, Hiatt WR (1988) Reduction of polygalactur-

onase activity in tomato fruit by antisense RNA. Proc Natl Acad

Sci USA 85:8805–8809

Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden

SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM,

Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D,

Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley

SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (2009) Precise

genome modification in the crop species Zea mays using zinc-

finger nucleases. Nature 459:437–441

Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F

(2016) Rationally engineered Cas9 nucleases with improved

specificity. Science 351:84–88

Sprink T, Metje J, Hartung F (2015) Plant genome editing by novel

tools: TALEN and other sequence specific nucleases. Curr Opin

Biotechnol 32:47–53

Srivastava V, Gidoni D (2010) Site-specific gene integration

technologies for crop improvement. In Vitro Cell Dev Biol-

Plant 46:219–232

Srivastava V, Ow DW (2002) Biolistic mediated site-specific

integration in rice. Mol Breeding 8:345–350

Srivastava V, Ow DW (2004) Marker-free site-specific gene integra-

tion in plants. Trends Biotechnol 22:627–629

Srivastava V, Thomson J (2016) Gene stacking by recombinases.

Plant Biotechnol J 14:471–482

Srivastava V, Ariza-Nieto M, Wilson AJ (2004) Cre-mediated site-

specific gene integration for consistent transgene expression in

rice. Plant Biotechnol J 2:169–179

Steinert J, Schiml S, Fauser F, Puchta H (2015) Highly efficient

heritable plant genome engineering using Cas9 orthologues from

Streptococcus thermophilus and Staphylococcus aureus. Plant J

84:1295–1305

Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H, Xi Y (2015)

Targeted mutagenesis in soybean using the CRISPR-Cas9

system. Sci Rep 5:10342

Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of

plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530

Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM

(2015) Targeted mutagenesis, precise gene editing, and site-

specific gene insertion in maize using Cas9 and guide RNA.

Plant Physiol 169:931–945

Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S (2002) Efficient

gene targeting by homologous recombination in rice. Nat

Biotechnol 20:1030–1034

Terada R, Johzuka-Hisatomi Y, Saitoh M, Asao H, Iida S (2007) Gene

targeting by homologous recombination as a biotechnological tool

for rice functional genomics. Plant Physiol 144:846–856

Terada R, Nagahara M, Furukawa K, Shimamoto M, Yamaguchi K,

Iida S (2010) Cre-loxP mediated marker elimination and gene

reactivation at the waxy locus created in rice genome based on

strong positive-negative selection. Plant Biotechnol 27:29–37

Thomson JG, Blechl A (2015) Recombinase technology for precise

genome engineering. In: Zhang F, Puchta H, Thomson JG (eds)

Advances in new technology for targeted modification of plant

genomes. Springer, New York, pp 113–144

Thykjaer T, Finnemann J, Schauser L, Christensen L, Poulsen C,

Stougaard J (1997) Gene targeting approaches using positive-

negative selection and large flanking regions. Plant Mol Biol

35:523–530

Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK,

Voytas DF (2009) High-frequency modification of plant genes

using engineered zinc-finger nucleases. Nature 459:442–445

Tzfira T, Frankman LR, Vaidya M, Citovsky V (2003) Site-specific

integration of Agrobacterium tumefaciens T-DNA via double-

stranded intermediates. Plant Physiol 133:1011–1023

van der Steege G, Schuilenga-Hut PHL, Buys C, Scheffer H, Pas HH,

Jonkman MF (2001) Persistent failures in gene repair. Nat

Biotechnol 19:305–306

Vergunst AC, Hooykaas PJJ (1998) Cre/lox-mediated site-specific

integration of Agrobacterium T-DNA in Arabidopsis thaliana by

transient expression of cre. Plant Mol Biol 38:393–406

Vergunst AC, Jansen LET, Hooykaas PJJ (1998) Site-specific

integration of Agrobacterium T-DNA in Arabidopsis thaliana

mediated by Cre recombinase. Nucleic Acids Res 26:2729–2734

Voytas DF (2013) Plant genome engineering with sequence-specific

nucleases. Annu Rev Plant Biol 64:327–350

Plant Cell Rep (2016) 35:1401–1416 1415

123

Wakasa Y, Hayashi S, Ozawa K, Takaiwa F (2012) Multiple roles of

the ER stress sensor IRE1 demonstrated by gene targeting in

rice. Sci Rep 2:944

Wang Y, Yau YY, Perkins-Balding D, Thomson JG (2011) Recom-

binase technology: applications and possibilities. Plant Cell Rep

30:267–285

Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL (2014)

Simultaneous editing of three homoeoalleles in hexaploid bread

wheat confers heritable resistance to powdery mildew. Nat

Biotechnol 32:947–951

Wang M, Liu Y, Zhang C, Liu J, Liu X, Wang L, Wang W, Chen H,

Wei C, Ye X, Li X, Tu J (2015a) Gene editing by co-

transformation of TALEN and chimeric RNA/DNA oligonu-

cleotides on the rice OsEPSPS gene and the inheritance of

mutations. PLoS One 10:e0122755

Wang S, Zhang S, Wang W, Xiong X, Meng F, Cui X (2015b)

Efficient targeted mutagenesis in potato by the CRISPR/Cas9

system. Plant Cell Rep 34:1473–1476

Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen

QJ (2015c) Egg cell-specific promoter-controlled CRISPR/Cas9

efficiently generates homozygous mutants for multiple target

genes in Arabidopsis in a single generation. Genome Biol 16:144

Weeks DP, Spalding MH, Yang B (2016) Use of designer nucleases

for targeted gene and genome editing in plants. Plant Biotechnol

J 14:483–495

Wendt T, Holm PB, Starker CG, Christian M, Voytas DF, Brinch-

Pedersen H, Holme IB (2013) TAL effector nucleases induce

mutations at a pre-selected location in the genome of primary

barley transformants. Plant Mol Biol 83:279–285

Woo JW, Kim J, Kwon SI, Corvalan C, Cho SW, Kim H, Kim SG,

Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in

plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat

Biotechnol 33:1162–1164

Wright DA, Townsend JA, Winfrey RJ, Irwin PA, Rajagopal J,

Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) High-

frequency homologous recombination in plants mediated by

zinc-finger nucleases. Plant J 44:693–705

Xiaohui Wang H, Viret JF, Eldridge A, Perera R, Signer ER,

Chiurazzi M (2001) Positive-negative selection for homologous

recombination in Arabidopsis. Gene 272:249–255

Xie K, Zhang J, Yang Y (2014) Genome-wide prediction of highly

specific guide RNA spacers for CRISPR–Cas9-mediated genome

editing in model plants and major crops. Mol Plant 7:923–926

Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9

multiplex editing capability with the endogenous tRNA-process-

ing system. Proc Natl Acad Sci USA 112:3570–3575

Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC,

Chen QJ (2014) A CRISPR/Cas9 toolkit for multiplex genome

editing in plants. BMC Plant Biol 14:327

Xu Y (2010) Molecular plant breeding. CAB International,

Wallingford

Xu R, Li H, Qin R, Wang L, Li L, Wei P, Yang J (2014) Gene

targeting using the Agrobacterium tumefaciens-mediated

CRISPR-Cas system in rice. Rice (NY) 7:5

Yamauchi T, Iida S (2015) Gene targeting in crop species with

effective selection systems. In: Zhang F, Puchta H, Thomson JG

(eds) Advances in new technology for targeted modification of

plant genomes. Springer, New York, pp 91–111

Yamauchi T, Johzuka-Hisatomi Y, Fukada-Tanaka S, Terada R,

Nakamura I, Iida S (2009) Homologous recombination-mediated

knock-in targeting of the MET1a gene for a maintenance DNA

methyltransferase reproducibly reveals dosage-dependent spa-

tiotemporal gene expression in rice. Plant J 60:386–396

Yamauchi T, Johzuka-Hisatomi Y, Terada R, Nakamura I, Iida S

(2014) The MET1b gene encoding a maintenance DNA

methyltransferase is indispensable for normal development in

rice. Plant Mol Biol 85:219–232

Yan L, Wei S, Wu Y, Hu R, Li H, Yang W, Xie Q (2015) High-

efficiency genome editing in Arabidopsis using YAO promoter-

driven CRISPR/Cas9 system. Mol Plant 8:1820–1823

Yau Y-Y, Wang Y, Thomson JG, Ow DW (2011) Method for Bxb1-

mediated site-specific integration in planta. Methods Mol Biol

701:147–166

Yin K, Han T, Liu G, Chen T, Wang Y, Yu AY, Liu Y (2015) A

geminivirus-based guide RNA delivery system for CRISPR/Cas9

mediated plant genome editing. Sci Rep 5:14926

Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova

KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A,

Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided

endonuclease of a class 2 CRISPR-Cas system. Cell

163:759–771

Zhang F, Maeder ML, Unger-Wallace E, Hoshaw JP, Reyon D,

Christian M, Li X, Pierick CJ, Dobbs D, Peterson T, Joung JK,

Voytas DF (2010) High frequency targeted mutagenesis in

Arabidopsis thaliana using zinc finger nucleases. Proc Natl Acad

Sci USA 107:12028–12033

Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ,

Voytas DF (2013) Transcription activator-like effector nucleases

enable efficient plant genome engineering. Plant Physiol 161:20–27

Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L,

Zhang H, Xu N, Zhu JK (2014) The CRISPR/Cas9 system

produces specific and homozygous targeted gene editing in rice

in one generation. Plant Biotechnol J 12:797–807

Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large

chromosomal deletions and heritable small genetic changes

induced by CRISPR/Cas9 in rice. Nucleic Acids Res

42:10903–10914

Zhou J, Peng Z, Long J, Sosso D, Liu B, Eom J-S, Huang S, Liu S,

Vera Cruz C, Frommer WB, White FF, Yang B (2015a) Gene

targeting by the TAL effector PthXo2 reveals cryptic resistance

gene for bacterial blight of rice. Plant J 82:632–643

Zhou X, Jacobs TB, Xue L-J, Harding SA, Tsai C-J (2015b)

Exploiting SNPs for biallelic CRISPR mutations in the outcross-

ing woody perennial Populus reveals 4-coumarate: CoA ligase

specificity and redundancy. New Phytol 208:298–301

Zhu T, Peterson DJ, Tagliani L, St Clair G, Baszczynski CL, Bowen

B (1999) Targeted manipulation of maize genes in vivo using

chimeric RNA/DNA oligonucleotides. Proc Natl Acad Sci USA

96:8768–8773

Zhu T, Mettenburg K, Peterson DJ, Tagliani L, Baszczynski CL

(2000) Engineering herbicide-resistant maize using chimeric

RNA/DNA oligonucleotides. Nat Biotechnol 18:555–558

1416 Plant Cell Rep (2016) 35:1401–1416

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