Download Site-specific recombination for genetic engineering in plants

Plant Cell Rep (2003) 21:925–932
DOI 10.1007/s00299-003-0616-7
REVIEW
L. A. Lyznik · W. J. Gordon-Kamm · Y. Tao
Site-specific recombination for genetic engineering in plants
Received: 20 December 2002 / Revised: 19 February 2003 / Accepted: 24 February 2003 / Published online: 26 April 2003
Springer-Verlag 2003
Abstract Site-specific recombination has been developed into a genetic engineering tool for higher eukaryotes. The manipulation of newly introduced DNA is now
possible in the course of genetic transformation procedures, thus making the process more predictable and
reliable. Also, a wide variety of chromosomal rearrangements using site-specific recombination have been documented both in metazoan and plant species. Applying
such methods to plants opens new avenues for large-scale
chromosome engineering in the future.
Keywords Site-specific recombination · Plant
transformation · Genetic engineering · FLP/FRT ·
Cre/loxP
Abbreviations Cre: Causes recombination ·
FLP: Flipping DNA · FRT: FLP recombination target ·
loxP: Locus of crossing over in P1
Introduction
For many crop species, transformation has become
routine. But as the technology has advanced, the expectations for transgenic crops are becoming more demanding. Precision and predictability in the production of
transgenic crops will become increasingly important. One
step toward increasing the quality of transgenic events is
the shift toward using Agrobacterium (as opposed to
physical methods of DNA delivery) in all crops where this
is practical. The advantages of Agrobacterium such as
low transgene copy number (Hobbs et al. 1993; Ingham et
al. 2001) and preferential integration into actively transcribed locations (Szabados et al. 2002) have been well
Communicated by P.P. Kumar
L. A. Lyznik ()) · W. J. Gordon-Kamm · Y. Tao
Transformation Research, Pioneer Hi-Bred International Inc.,
7300 N.W. 62nd Avenue, Johnston, IA 50131, USA
e-mail: [email protected]
Tel.: +1-515-254-2674
Fax: +1-515-270-3444
documented, and are now being realized in many cereal
crops thought to be recalcitrant to this method just a few
years ago (Komari and Kubo 1999). Yet, despite the
benefits of using Agrobacterium, integration is still
random, contributing to variable expression between
transgenic events and offering no ability to control the
impact of genomic position on transgene silencing.
How can we take the next step to increase the precision
of transgene integration and potentially improve the
stability of gene expression? The combination of homologous and site-specific recombination has emerged as a
powerful chromosome-engineering technology in mammals and insects, in particular, mouse embryonic stem
cells and Drosophila (Rong et al. 2002; Yu and Bradley
2001). In the most common scenario, foreign DNA
fragments are integrated into predetermined chromosomal
locations by homologous recombination, and site-specific
recombination is used for subsequent modifications of
transgenic loci. Such technology offers an unprecedented
level of manipulation of genomic DNA sequences. The
Cre/loxP and FLP/FRT systems (named as follows: Cre
causes recombination; loxP locus of crossing over in P1;
FLP flipping DNA; FRT FLP recombination target)
provide site-specific recombination activity for such
applications. While we are not yet at the point of
combining homologous and site-specific recombination
methods in plants, both FLP and Cre have been used to
integrate new transgenes into pre-existing transgenic loci
in the genome (Day et al. 2000; Ow 2002), demonstrating
that precise transgene placement is feasible. But clever
use of recombinases also confers flexibility, permitting a
variety of controlled genetic modifications, including
excisions, inversions and translocations. The Cre/loxP
system has even been used to create hybrid chromosomes,
recombining a small portion of an Arabidopsis chromosome into the tobacco genome (Koshinsky et al. 2000). As
yet, examples of such FLP- or Cre-mediated processes in
plants are still not commonplace, but progress in this area
is accelerating. As recombinase-based methods continue
to build on the current technology, their use in precise
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transgenic applications will become increasingly important for crop biotechnology.
Site-specific recombination systems
Site-specific recombination systems are common in
prokaryotes and lower eukaryotes. Natural functions of
such systems include integration of a bacteriophage into
the host genome (bacteriophage l, Streptomyces phage
jC31, lactococcal bacteriophage TP901-1), maintenance
of copy number (the Cre/loxP system of bacteriophage
P1), and switching host range (the Gin/gix system of
bacteriophage Mu). In addition, transposons may use sitespecific recombination for generation of the final products
of transpositions (class II transposable elements such as
Tn3). Autonomous plasmid DNA molecules may invert
their convergent replication forks to accelerate the
replication process (the FLP/FRT system of yeast), and
bacteria may use site-specific recombination for altering
cell-surface components (for example, the Hin system of
Salmonella typhimurium). The examples described here
represent a few of the hundreds of site-specific recombination systems that are categorized as integrases,
resolvases, and invertases (Esposito and Scocca 1997).
Within this large group of enzymes, the complexity and
specificity of the recombination reaction varies within
different families of recombination systems. However,
resolvases and invertases are generally more selective
than integrases (Nunes-Duby et al. 1998).
In this review, we focus our attention on site-specific
recombination systems that have been most widely used
in plants, in particular the FLP/FRT and Cre/loxP systems
from yeast and bacteriophage P1, respectively (Odell and
Russell 1994). They catalyze conservative, reciprocal
exchanges of DNA strands at specific DNA sites. During
this exchange, the phosphodiester bonds within the
recombination site are broken by nucleophilic attack of
hydroxyl groups originating from a tyrosine residue
residing within the C-terminal domain of the recombinase. In the sequential transesterification reactions, four
monomers participate in the formation of the recombination product (Guo et al. 1997; Voziyanov et al. 1999).
Resolvases and invertases efficiently mediate DNA
recombination in specifically arranged recombination
sites, e.g. two sites in the same orientation on the same
molecule are recombined efficiently by resolvases but not
invertases, which, in turn, require two sites in the opposite
orientation. Cre and FLP, however, are relatively simple
recombinases that do not discriminate topological features
between substrates entering the recombination pathway.
For tyrosine recombinases such as FLP and Cre, the
orientation of the recombination sites relative to each
other can determine the outcome of the reaction. Direct
orientation of two recombination sites in one DNA
molecule leads to intramolecular recombination or excision, leaving behind a recombination site as a footprint on
the parent molecule. A second circularized DNA fragment containing the other recombination site is also
Fig. 1A–C Site-specific recombination reactions. Since FLP/FRT
and Cre/loxP are fully reversible reactions, the outcome depends on
the placement of the recombination sites and their relative
orientation. A, B Single-site integration events may be stabilized
by limiting the availability of recombinases, while double-reciprocal crossover is stabilized by elimination of the substrate. C
Relatively short DNA sequences, FRT and loxP, are the DNA
binding sites for FLP and Cre, respectively. Asymmetry of the
spacer region (underlined) determines the site orientation. Mutations within the spacer region (marked in red), as exemplified by
the FTR5 site (Schlake and Bode 1994), render the sites incompatible with each other but still functional, at least partially (see
Fig. 4)
produced (Fig. 1A). The reverse reaction, when the
recombination sites are located on two DNA molecules,
produces an intermolecular recombination or integration.
When two pairs of recombination sites are involved in
intermolecular recombination, the double recombination
events lead to exchange of a DNA fragment delimited by
the recombination sites (Fig. 1B).
Simple, site-specific recombination systems
are functional in plant cells
The first wave of practical applications of site-specific
recombination systems for genetic studies in plants used
relatively simple, two-component systems such as Cre/
loxP from the bacteriophage P1, FLP/FRT or R/RS from
yeast, or Gin/gix from bacteriophage Mu (Odell and
Russell 1994). Initial studies utilized transient expression
assays in plant protoplasts in order to demonstrate
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functionality of Cre in tobacco (Dale and Ow 1990), R in
tobacco (Onouchi et al. 1991), FLP in maize and rice
(Lyznik et al. 1993), and Gin in Arabidopsis and tobacco
(Maeser and Kahmann 1991). Subsequently, this work
was extended to chromosomal excision of DNA fragments flanked by recombination sites (Dale and Ow 1991;
Lyznik et al. 1996; Odell et al. 1990; Onouchi et al.
1995). Thus, the systems were proven to be reliable and
efficient in performing chromosomal DNA recombination. They have been used successfully for the removal of
selectable markers from nuclear chloroplasts and DNA in
transgenic plants (Corneille et al. 2001; Gleave et al.
1999; Lloyd and Davis 1994; Sugita et al. 2000; Zuo et al.
2001). The Cre/loxP recombination system was used to
reduce the copy number of DNA insertions in transgenic
wheat and maize plants after bombardment (Srivastava
and Ow 2001a, b; Srivastava et al. 1999). It has been
shown that multiple copies of a transgene may lead to
silencing of expression (De Buck et al. 2001), and thus it
is not surprising that Cre-mediated removal of silencing
sequences preserves stability of transgene expression (De
Buck and Depicker 2001). Recently, Cre/loxP function
was also demonstrated in rice, extending such possible
applications into yet another important crop plant (Hoa et
al. 2002; Srivastava and Ow 2001a, b).
The most straightforward approach to site-specific
recombination in plant cells is the removal of undesired
DNA sequences (such as selectable markers) from
transformation vectors. First, a DNA cassette containing
the recombination sites is randomly integrated into a
genomic DNA and then another round of transformation
(re-transformation) is performed with a recombinaseexpression vector. Genomic excisions can be identified at
a 3–5% overall efficiency in maize protoplasts (no
selection for the recombination or re-transformation
events) (Hodges and Lyznik 1996). The same strategy
produces 0.25% excision efficiency in Agrobacteriummediated transient transformation of tobacco cells with
Cre (Gleave et al. 1999). For production of transgenic
crop plants, the process may not be practical though.
Transgenic event production time is doubled, the exposure to tissue-culture conditions is prolonged, and it may
still be necessary to apply a segregation step in order to
remove the recombinase gene from the final product. In
another approach, the recombinase gene can be introduced by crossing two parental transgenic plants, one of
them containing the recombination substrate. This approach was first demonstrated in tobacco using Cre/loxP
(Odell et al. 1990). The recombination seems to be almost
100% efficient (Fig. 2) (Lloyd and Davis 1994). However,
germline inheritance of recombined loci may require a
strong expression of recombinase early after zygote
formation (Gidoni et al. 2001). If the recombinasecontaining parental plant is established as a final,
desirable genotype for post-transformation backcrossing
activities, this procedure does not necessarily add additional steps in the production of transgenic plants.
However, segregation of two transgenic traits (the
recombinase gene and the original transgene) may still
Fig. 2 A sample of maize seedlings tested for FLP-mediated
excision of a recombination substrate. Two transgenic parental
maize plants were crossed (homozygous FLP-expressing plant excision-substrate containing To plant) and embryos were rescued
15 days after pollination. After germination, samples of leaf tissue
were used for DNA extraction and PCR analysis. Fifty percent of
embryos were expected to contain both the FLP gene and the
recombination substrate. Thirty six out of 80 samples showed clear
evidence of excision, i.e. they showed the expected band after PCR.
The white arrow points to the positive-control band
be required. Recently, Zuo et al. (2001) designed an
elegant one-step transformation strategy taking advantage
of an effective, estrogen-regulated Cre induction system.
Upon induced DNA excision, both the selectable marker
gene and the Cre recombinase gene (self-excision) were
removed. Unlike crossing two transgenic parental plants
or re-transformation, this is indeed a one step procedure.
Site-specific recombination can be used
to integrate foreign DNA into plant genomic DNA
Site-specific recombination may offer a solution to a
fundamental predicament of genetic engineering in plants:
how to make the process efficient, predictable, and
reliable. The current status of plant transformation
technology is far from being considered true “genetic
engineering”. Even using Agrobacterium, the most efficient random transformation system available, hundreds
of original transformation events must often be evaluated
to find a commercially viable event conferring good
transgene expression and uncompromised agronomic
performance. This adds considerable time and cost to
produce acceptable transgenic crop plants. To potentially
reduce the variability in the process, Vergunst et al.
(1988) envisioned using Cre/loxP to integrate T-DNA
into pre-determined chromosomal loci of Arabidopsis.
Two approaches were taken to enable the formation of
substrate T-DNA within plant cells (the circular T-DNA
equivalent to the integration substrate shown in Fig. 1A).
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One was via border recombination in which a single loxP
site was placed in the T-DNA. The other was via
intramolecular recombination within the T-DNA containing two loxP sites, the loxP-35S-ATG-loxP cassette.
Unfortunately, the system was found to be only about 1–
2% efficient compared to random integration of T-DNAs.
In addition, a substantial number of recovered transformation events showed multiple chromosomal rearrangements.
These somewhat ambiguous results pose the question
whether T-DNA molecules are suitable substrates for sitespecific recombination (Vergunst and Hooykaas 1999).
Site-specific recombinases are, in fact, proteins that bind
double-stranded DNA, while T-DNA arrives into the
plant nucleus in a single-stranded DNA form, coated with
proteins such as virE (Zupan and Zambryski 1995) that
bind single-stranded DNA. T-DNAs probably do not need
to become double-stranded for integration to take place
(Mayerhofer et al. 1991). The limited availability of
double-stranded T-DNA substrate for site-specific recombination can be addressed by replicating T-DNA in
transformed plant cells (Zhao et al. 2003). Once T-DNA
is amplified, the majority of recovered T-DNA molecules
from maize cells are the products of site-specific recombination, if the components of this system are provided on
the T-DNA. At present, direct DNA delivery methods
seem to be a better choice for locus-specific integration of
foreign DNA. For example, PEG-mediated protoplast
transformation of tobacco was originally used to assess
the potential of the Cre/loxP system by integrating the
incoming plasmid into a single genomic recombination
site (Albert et al. 1995). The kinetics of this reversible
recombination reaction favor excision over integration,
so, without further modifications, single site integration
would be extremely inefficient (Baer and Bode 2001). To
overcome such unfavorable kinetics of site-specific
integration.
New strategies were developed. Two loxP sites, each
mutated in the opposite repeat sequence (one in the left
repeat and the other in the right repeat), were synthesized.
Site-specific recombination between the two different
single-mutation loxP sites still took place producing a
substantially less reactive double mutant (thus inhibiting
re-excision). The method was found feasible in tobacco
and was also applied to produce site-specific integrations
in mouse embryonic stem cells (Araki et al. 1997).
Another strategy that has proven effective at increasing
integration at a single-site locus relies on inactivation of
recombinase expression as a result of integration. For
example, Day et al. (2000) and Choi et al. (2000) used
such a strategy to demonstrate integration into a 35SloxP-Cre site in tobacco and Arabidopsis, respectively.
Choi et al. (2000) reported site-specific integration of
BAC clones up to 230 kb in length containing a
promoterless loxP-npt into the Arabidopsis genome,
while Day et al. (2000) used the system to test whether
chromosome position can affect the level of gene
expression in tobacco. The third strategy, not yet used
in plants, is to provide inducible expression of recombi-
Fig. 3A, B Relative recombination efficiency of FRT1 and FRT5
sites (see Fig. 1 for the spacer sequence). In an in vitro FLPmediated recombination assay shown in B, the recombination rate
of FRT1 sites is set for 100% (lane 1). Only a fraction of the FRT5containing substrate is recombined under the same conditions (lane
5), while the recombination between FRT1 and FRT5 is not
detected (lane 1+5)
nase. When FLP expression is stringently regulated, for
example under the control of the MAL2 promoter in
Candida albicans, genome-resident FRT sites are targeted
at high rates (Sanchez-Martinez and Perez-Martin 2002).
This remains a promising option for plant transformation,
since regulated expression of FLP and Cre has been
documented in Arabidopsis, for example, in the context of
studying flower development by excisional activation of
transcription factors (Hoff et al. 2001; Kilby et al. 2000;
Riou-Khamlichi et al. 1999; Sessions et al. 2000). While
such single-site strategies have clearly worked for
recombinase-mediated integration, further optimization
may continue to be hampered by the fact that excision
remains the favored reaction.
To obviate the problem of re-excision, alternative
methods have been developed using two independent
(incompatible) recombination sites (Bode et al. 2000). In
this method referred to as recombinase-mediated cassette
exchange (RMCE), a DNA fragment to be integrated is
placed between two incompatible recombination sites, for
example, FRT1 and FRT5, and is delivered into a preengineered genomic site containing these respective sites
(Figs. 1, 3). Starting with such substrates, FLP-mediated
recombination results in double reciprocal crossover,
exchanging a DNA fragment in the plasmid with the DNA
fragment between FRT1 and FRT5 in the genome. The
strategy was envisioned and tested in mammalian cells
(Schlake and Bode 1994; Seibler and Bode 1997), based
on the observation that, in simple recombination systems
such as Cre/loxP and FLP/FRT, the spacer region in the
recombination sites can be altered to change specificity of
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recombination sites (Lee and Saito 1998). The functionality of the system for plant transformation has been
documented in maize (Baszczynski et al. 2002). In this
case, GUS and a selectable marker gene replaced two
originally integrated transgenes (GFP and another selectable marker gene).
The RMCE strategy offers several advantages over
single-site integration: (1) the overall efficiency of sitespecific integration is increased by stabilizing the integration product (Feng et al. 1999); (2) only well-defined
DNA fragments flanked by two recombination sites are
integrated into a desired locus; (3) selection markers do
not need to be integrated into the final recombination
product (Kolb 2001); and (4) the method can be combined
with a negative selection scheme allowing selection
against random integrations (Terada et al. 2002). In
addition, multiple sites can be utilized to place multiple
trait genes into the same locus, a process called trait
stacking (Sauer 1996).
It seems, however, that the basic issue of efficiency
still needs to be addressed. It has been anticipated that
site-specific integration should be even more efficient
than random, accidental integration of foreign DNA.
Indeed, the RMCE strategy produced a 3-fold increase in
site-specific over random integrations in mouse NIH 3T3
cells (Bethke and Sauer 1997). Further refinement of this
procedure led to a consistent replacement of the loxdelimited genomic segment at the MHL-1 locus without
relying on any type of selection (Soukharev et al. 1999).
The method was further refined by Feng et al. (1999).
Using a new, well-tested mutant site lox2722, Kolb
(2001) was able to modify the murine b-casein gene with
90% efficiency. However, a few laboratories have
reported difficulties in implementing RCME type methods. This may be due to the fact that different lox sites can
still recombine with each other, while extensive mutagenesis of the spacer region compromises recombination
rates (Lee and Saito 1998). As an alternative solution, the
simultaneous use of two recombinases, FLP and Cre,
acting in concert on their respective sites, FRT and loxP,
may improve the efficiency of the RMCE strategy (Lauth
et al. 2002). The recombination substrate used for the
experiment presented in Fig. 4 indeed contained a
combination of FRT and loxP sites for excision and
subsequent integration of the recombination vector.
Protein engineering has been applied to improve the
overall performance of site-specific-recombination systems. Examples include increasing the temperature optimum for FLP-mediated recombination to 37C, making
FLP more effective in mammalian cells (Buchholz et al.
1998) and relaxing the substrate specificity of Cre to
favor recombination between lox-like sites (loxH) in
mouse cells (Buchholz and Stewart 2001). DNA shuffling
of the jC31 integrase has also produced a more integration-efficient version of this protein (Sclimenti et al.
2001). Further advances in recombinase engineering will
undoubtedly occur in the near future, aided by the
increasingly sophisticated methods of protein modeling
and engineering.
Fig. 4A–D Progeny of a cross between two transgenic maize plants
containing the FLP/Cre expression cassette and the recombination
substrate. A Excision of the substrate vectors activates a cyan
fluorescent marker. B Their integration generates yellow fluorescence (scattered yellow spots on a scutellar tissue). C, D When
relocation and integration of the recombination substrate take place
early during embryo development, sectored embryos emerge (a
two-cell-layered streak of yellow fluorescence across coleoptile)
Chromosome engineering using site-specific
recombination systems
In the future, site-specific recombination will contribute
to increasing the scope of DNA manipulation in eukaryotes. Genomic technologies are producing a wealth of
genetic information and along with this come an
increased need for physical, precise methods to manipulate chromosomal DNA molecules in vivo.
Large-scale plant chromosomal rearrangement using
the Cre/loxP system was originally reported for tobacco
chromosomes (Qin et al. 1994). When two parental
transgenic plants were crossed, the chromosomal translocation product (activated hygromycin-resistance gene)
was found in 2.4% of the seeds and 50% of the seedlings
expected to harbor both loxP and Cre loci. Four out of 16
combinations of parental plants produced chromosomal
translocations. Among them, co- segregation (pseudolinkage) of transgenic loci was observed indicating deleterious duplications and translocations in gametes or zygotes.
Medberry et al. (1995) and Osborne et al. (1995) reported
an interesting variation of this method. Two loxP sites
were integrated into a tobacco chromosome, one of them
within the non-autonomous Ds element. Two subsequent
crosses were conducted: one with parental plant containing the maize Ac transposon to relocate one loxP site, and
the subsequent cross with a transgenic plant expressing
the Cre recombinase to produce intra-chromosomal
rearrangement such as deletions and inversions (Ds
element tends to move close to the original insertion)(Medberry et al. 1995). A similar procedure produced inversions on Arabidopsis chromosome 4, one of
which was germinally transmitted from generation to
930
generation (Osborne et al. 1995). Such rearranged chromosomal fragments have been recovered using in vitro
site-specific recombination. Cre/loxP recombination between chromosomal loxP sites [generated by a Ds(lox)
transposable element in tomato] and synthetic loxP
oligonucleotides led to the release of 65 kb and 130 kb
fragments of chromosome 6 (Stuurman et al. 1996).
Inversion of the 130 kb fragment was also observed in
Cre-expressing tomato plants.
Plant chromosomal rearrangements were taken one
step further when tobacco and Arabidopsis protoplasts
were fused together in order to observe the formation of
hybrid chromosomes induced by the Cre/loxP recombination system (Koshinsky et al. 2000). A small portion of
Arabidopsis chromosome V was detected in the tobacco
genome after selection for hygromycin-resistant progeny
(a site-specific recombination product) of plants regenerated from fused protoplasts.
In some cases it has been observed that inter- and
intra-chromosomal site-specific recombination is so efficient that they pose a nuisance in other types of
transformation experiments. For example, Vergunst et
al. (2000) attempted site-specific integration of a plox-npt
T-DNA molecule into the 35S-lox-cre transgenic locus in
Arabidopsis. Ninety six percent of selected kanamycinresistant events were classified as chromosomal translocations, inversions, and deletions. Assuming a 1%
transformation efficiency compared with random integration of the same T-DNA molecules, site-specific integration of T-DNA occurred at a rate of 0.04% relative to
random integration, while inter- and/or intra-chromosomal site-specific recombination between randomly integrated T-DNAs was about 20-fold more prevalent.
Experimental quirks aside, chromosomal rearrangement induced by site-specific recombination is feasible
and practical in plants. This is an emerging technology
that, without doubt, will be converted into a highly
sophisticated chromosome-engineering tool as is already
being realized in mammals (Yu and Bradley 2001).
Future prospects
The use of recombinase systems in plants has for many
years been seen as being analogous to clever parlor tricks,
but recently their value and utility have been receiving
more serious attention. The use of recombinases for
transgene excision or resolving complex transgenic loci
will likely become common tools in the production of
transgenic products. But we predict that a major impact of
recombinase technology in the production of transgenics
will come from continued improvements in site-specific
integration. Precise integration of new genes into predetermined and well-characterized genomic sites would
have some clear advantages. The recombination catalyzed
by these enzymes occurs with high fidelity (for all
practical purposes the reaction is perfect), and this
precision may ultimately help in the areas of regulatory
costs and public approval. Being able to introduce genes
reproducibly into a chosen location will also allow us to
collect valuable information on the role of position effect
in transgene silencing, and may help us find ways to
remedy this problem. The method will also allow us to
identify useful target sites in the genome that both confer
reasonable transgene expression and display no agronomic yield drag.
Finally, recombinase systems may help us take
advantage of our increased understanding of plant
genome structure to undertake chromosome engineering
projects. Such methods will likely augment plant breeding
programs, permitting not only inter- and intra-chromosomal engineering within the same species, but also
providing methods for moving genomic fragments between related species. Such large-scale modifications may
offer new possibilities in such areas as wide hybridization
research.
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