RESEARCH ARTICLES
DNA Replicons for Plant Genome EngineeringW OPEN
Nicholas J. Baltes, Javier Gil-Humanes, Tomas Cermak, Paul A. Atkins, and Daniel F. Voytas1
Department of Genetics, Cell Biology, and Development, Center for Genome Engineering, University of Minnesota, Minneapolis,
Minnesota 55455
Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant
genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use
geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator–
like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair
templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one
to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA doublestrand
breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication
initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate plants with a desired DNA sequence
modification. By adopting a general plant transformation method, plantlets with a desired DNA change were regenerated in <6 weeks.
These results, in addition to the large host range of geminiviruses, advocate the use of replicons for plant genome engineering.
INTRODUCTION
Precise modification of plant DNA is of value for basic and applied
biology because it facilitates gene function studies and crop
improvement. One method to modify DNA involves introducing
targeted double-strand breaks. Double-strand breaks activate
two main repair pathways: nonhomologous end joining (NHEJ)
and homologous recombination. These two pathways can be
exploited to introduce sequence changes within the plant genome.
Repair by NHEJ is often imprecise, leading to insertions or
deletions of DNA at the cut site. Consequently, double-strand
breaks directed to endogenous genes can facilitate targeted gene
disruption or targeted mutagenesis (Lloyd et al., 2005; Zhang
et al., 2010). Double-strand breaks can also be repaired with high
fidelity by homologous recombination. Homologous recombination
normally involves the use of the sister chromatid or homologous
chromosome for template-directed repair. If an exogenously supplied
repair template is present, gene replacement or targeted gene
insertion can be performed (referred to hereafter as gene targeting;
Puchta et al., 1996; Bibikova et al., 2003).
Targeted double-strand breaks can be generated using sequencespecific
nucleases. These nucleases include meganucleases (Smith
et al., 2006), zinc-finger nucleases (ZFNs; Kim et al., 1996),
transcription activator–like effector nucleases (TALENs; Christian
et al., 2010; Miller et al., 2011), and the clustered, regularly interspaced,
short palindromic repeat (CRISPR)–associated
protein (Cas) system (Cong et al., 2013; Mali et al., 2013). Using
sequence-specific nucleases, plant genomes have been modified
in many different ways, ranging from several nucleotide
substitutions (Townsend et al., 2009) to the targeted deletion of
megabases of DNA (Qi et al., 2013).
In plants, delivery of genome engineering reagents, including
sequence-specific nucleases and repair templates, presents a barrier
to efficiently achieving targeted genome modifications. For gene
targeting, there are a few reports using Agrobacterium tumefaciens
or physical means to deliver genome engineering reagents (Cai
et al., 2009; Shukla et al., 2009); however, the highest frequencies of
gene targeting have been achieved using protoplasts, plant cells
lacking cell walls (Wright et al., 2005; Townsend et al., 2009; Zhang
et al., 2010). Protoplasts can be transformed at high efficiency, but
only a handful of plants can be effectively regenerated from protoplasts.
Alternatively, constructs encoding nucleases and repair
templates can be integrated into plant genomes (Fauser et al., 2012;
Ayar et al., 2013). This so-called in planta genome engineering
strategy relies on the gene targeting to occur during the life of the
plant, with the anticipation that germline cells will be modified and
ultimately give rise to seed with the desired sequence alterations.
In mammalian systems, effective delivery of genome engineering
reagents has been achieved using viruses, including retroviruses
(Ellis and Bernstein, 1989; Lombardo et al., 2007),
adenoviruses (Wang and Taylor, 1993), and adeno-associated
viruses (Russell and Hirata, 1998). In plants, the RNA virus Tobacco
rattle virus was used to deliver a ZFN pair to petunia (Petunia
hybrida) and tobacco (Nicotiana tabacum) leaves, resulting in targeted
modification of an integrated reporter gene in somatic cells
(Marton et al., 2010). Whereas the cargo capacity of plant RNA
viruses enables delivery of a pair of ZFNs or a meganuclease, it
is not sufficient to deliver larger proteins, such as a pair of TALENs
or Cas9 for the CRISPR/Cas system. Furthermore, RNA viruses
cannot deliver DNA repair templates for gene targeting.
1 Address correspondence to voytas@umn.edu.
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Daniel F. Voytas (voytas@
umn.edu).
W
Online version contains Web-only data.
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www.plantcell.org/cgi/doi/10.1105/tpc.113.119792
The Plant Cell, Vol. 26: 151–163, January 2014, www.plantcell.org ã 2014 American Society of Plant Biologists. All rights reserved.
To develop an efficient and facile vector system for plant genome
engineering, we focused our efforts on the geminiviruses.
Geminiviruses are a large family of plant viruses with singlestranded,
circular DNA genomes of ;2.5 to 3.0 kb. Once inside
a host cell’s nucleus, their single-stranded genome is converted
to a double-stranded intermediate by host DNA polymerases.
This double-stranded genome is then used as a template for
transcription of virus genes and for rolling-circle replication. The
only geminivirus protein necessary for replication is the replication
initiator protein (Rep). Rep initiates rolling-circle replication
by binding to a site within the large intergenic region (LIR;
Mastreviruses), intergenic region (Curtoviruses and Topocuviruses),
or common region (Begomoviruses with bipartite genomes). There,
it creates a single-strand nick within an invariant 9-nucleotide sequence
(TAATATTAC) located at the apex of a conserved hairpin
structure. Following rolling-circle replication, single-stranded genomes
are either converted back to a double-stranded intermediate
or encapsidated by coat protein to produce virions. These virions
are then transported to adjacent cells through the plant’s
endogenous plasmodesmata pathways.
Methods to engineer geminiviruses for protein expression include
the full virus and deconstructed virus strategies (Gleba et al.,
2004). Under the full virus strategy, geminiviruses retain most or all
of the features required for replication and systemic infection of the
host plant. Heterologous sequence can be introduced within the
genome by replacing the coat protein gene (coat protein is not
required for cell-to-cell movement in some bipartite begomoviruses;
Gardiner et al., 1988). Using this approach, tomato golden
mosaic virus and African cassava mosaic virus were modified to
express neomycin phosphotransferase (Hayes et al., 1988) and
chloramphenicol acetyltransferase (Ward et al., 1988), respectively.
Unfortunately, it may not be possible to deliver relatively large
heterologous sequences while maintaining cell-to-cell movement
and rolling-circle replication. Cell-to-cell movement through the
endogenous plasmodesmata pathways imposes strict requirements
on the geminivirus genome size (Etessami et al., 1989;
Hayes et al., 1989; Elmer and Rogers, 1990; Gilbertson et al.,
2003). Larger than wild-type genomes frequently recombine to
smaller sizes and, as a result, are outcompeted during the infection
process. Whereas most studies exploring genome size restrictions
use bipartite begomoviruses, these constraints may also be experienced
with other geminiviruses, including mastreviruses. For
example, using the maize streak virus, ;3.2 kb of heterologous
sequence was inserted into the small intergenic region. Insertion of
sequence into this region does not disrupt virus replication or
expression of virus proteins (Shen and Hohn, 1991). After delivery
to leaf cells, the chimeric virus replicated efficiently; however, no
systemic movement was observed (Shen and Hohn, 1994).
Under the deconstructed virus strategy, limiting or undesired virus
functions are removed, and only the useful blocks are kept. For
example, to circumvent genome size constraints imposed by the
plasmodesmata, movement protein and coat protein sequences are
removed, whereas the cis- and trans-acting replicational elements
are retained. Lack of cell-to-cell movement can be compensated
using Agrobacterium or particle bombardment to deliver viral vectors
to cells. This approach has been successfully used to generate
replicons based on the bean yellow dwarf virus (BeYDV) for expression
of reporter proteins, vaccine proteins and monoclonal
antibodies (Mor et al., 2003; Zhang and Mason, 2006; Huang et al.,
2009, 2010; Regnard et al., 2010). Currently, there is no obvious
upper size limit for replicons that do not move from cell to cell (Laufs
et al., 1990; Matzeit et al., 1991; Shen and Hohn, 1994; Huang et al.,
2009). However, it may be that as replicon size increases, replication
efficiency decreases (Suárez-López and Gutiérrez, 1997).
The aim of this study was to explore the use of geminiviruses
for plant genome engineering. We demonstrate that DNA carried
by geminiviruses can be used as a template for homologous recombination.
We further demonstrate that geminivirus-based
replicons enable highly efficient genome engineering. We show
that this technology can be used to produce calli and plantlets with
precise DNA sequence changes. Finally, we develop a simplified
geminivirus vector system for efficient plant genome engineering.
RESULTS
Full Viruses for the Delivery of Repair Templates
in Arabidopsis
We began our study by exploring the use of full viruses for the
delivery of repair templates. Our target locus was the Arabidopsis
thaliana alcohol dehydrogenase (ADH1) gene. We modified the bipartite
cabbage leaf curl virus (CaLCuV) DNA A genome to contain
repair templates designed to introduce a unique 18-bp sequence
within ADH1 (Supplemental Figure 1A). After optimizing repair
template length (Supplemental Figures 1B to 1D), Arabidopsis
plants were infected with CaLCuV (harboring a repair template with
600 bp of total sequence) by particle bombardment (Supplemental
Figure 2). Within the genome of the infected plants was a ZFN pair
that targets a site within the ADH1 gene (Zhang et al., 2010). This
ZFN pair was under the transcriptional control of an estrogeninducible
promoter (Zuo et al., 2000). As a result, double-strand
breaks could be temporally controlled by spraying Arabidopsis
plants with a solution containing estradiol (Supplemental Figure 3).
Recombinant CaLCuV genomes were given 14 d to replicate and
spread before ZFN expression was induced. Seven days after
spraying, genomic DNA was isolated and the ADH1 gene was
assessed for the presence of the unique 18-bp sequence. From 23
experiments, we observed a single plant where the gene targeting
product could be detected by PCR (Supplemental Figure 4). While
these results suggest gene targeting with full viruses is possible,
further optimization of this technology was warranted.
Our approach using full viruses has several potential drawbacks,
including (1) the uncertainty of having both gene targeting reagents
(ZFN protein and geminivirus DNA) in a nucleus at the same time
and (2) the variability in the concentration of these reagents within
the same cell. For example, because we did not observe NHEJinduced
mutations within all ADH1 loci (Supplemental Figure 3),
we could not ensure that double-strand breaks were being produced
in all cells after spraying with estradiol. Furthermore, leaky
expression of the ZFN pair from the XVE promoter may result in
premature double-strand breaks and subsequent NHEJ mutations
that destroy the ZFN binding domain. Due to these uncertainties,
we sought to develop a system where (1) geminivirus vectors
could be efficiently delivered to target cells of interest and (2)
sequence-specific nucleases and repair templates are simultaneously
delivered.
152 The Plant Cell
Deconstructed Viruses for the Delivery of
Sequence-Specific Nucleases and Repair Templates
We chose to deconstruct the mild strain of the BeYDV (Halley-Stott
et al., 2007) because it had previously been used to express
heterologous proteins in plant cells (Regnard et al., 2010). BeYDV
replication requires three viral elements: the cis-acting LIR, the
short intergenic region (SIR), and the trans-acting replicationinitiation
protein (Rep) and RepA. Rep and RepA are expressed
from the same precursor mRNA: Rep is produced from the
spliced mRNA, whereas the unspliced mRNA yields RepA
(Wright et al., 1997). Eliminating coding sequences for the coat and
movement proteins abolishes cell-to-cell movement but effectively
eliminates genome-size constraints and makes it possible to
increase vector cargo capacity. To compensate for loss of cellto-cell
movement, we used Agrobacterium to deliver geminivirus
constructs to cells. The Agrobacterium T-DNA was modified to
harbor cis-acting sequences needed for replication in a LIR-SIRLIR
orientation (hereafter referred to as the LSL T-DNA; Figure
1A). Concomitant delivery of Rep protein should result in replicational
release and amplification of circular geminivirus replicons
(GVRs; Stenger et al., 1991).
Delivery of conventional T-DNA to plant cells by Agrobacterium
can result in transient gene expression. By modifying T-DNA to
harbor sequences for genome engineering reagents (sequencespecific
nucleases and repair templates), both targeted mutagenesis
and gene targeting can be performed (Reiss et al., 2000;
Li et al., 2013). To better distinguish between the effects of GVRs
and the input T-DNA, we sought to design the LSL T-DNA such
that expression of heterologous sequence is minimized when virus
proteins are not present. We chose to position heterologous
sequence downstream of the LIR in the virion-sense direction. The
LIR functions as a bidirectional promoter and promoter strength in
the virion-sense direction is weak in the absence of Rep and RepA
proteins (Hefferon et al., 2006). To promote high gene expression
within circularized GVRs, we positioned a duplicated 35S promoter
(P35S) upstream of the LIR in the virion-sense direction.
Splice donor and acceptor sequences were positioned upstream
and downstream, respectively, of the LIR sequence that is predicted
to form after circularization. High-protein expression may
also occur from the transactivation of the virion-sense LIR promoter
by the viral Rep proteins (Hefferon et al., 2006).
To investigate whether our GVRs replicate and express protein
in plant cells, we modified LSL T-DNA plasmid to encode green
fluorescent protein (GFP; pLSLGFP) or b-glucuronidase (GUS;
pLSLGUS; Figure 1B). GVRs were delivered to tobacco leaf tissue
by infiltrating a mixture of Agrobacterium strains containing
pLSLGFP or pLSLGUS and a separate T-DNA expression plasmid
encoding Rep and RepA (pREP). Both GUS and GFP expression
was enhanced when pREP T-DNA was delivered (Figure 1B;
Supplemental Figure 5). Background GUS activity seen in the tissue
transformed with pLSLGUS is most likely due to activity of the
virion-sense LIR promoter within Agrobacterium and plant cells.
To correlate the enhanced protein expression with GVR production,
replicational release was evaluated by PCR using primers
that would only amplify a product if circularization occurred.
Strong amplification of the LIR sequence was observed only from
samples in which pREP T-DNA was delivered, confirming successful
replicational release of replicons (Figure 1C; Supplemental
Figure 6). In a separate experiment, we quantified the relative GVR
gene copy number over a 2-week time course using quantitative
PCR (Supplemental Figure 7). Five days after infiltration, we
Figure 1. Development of GVRs for Protein Expression in Tobacco.
(A) Approach to deliver GVRs to plant cells. Rep is delivered using a separate T-DNA expression plasmid (pREP; data not shown). LB, left T-DNA
border; RB, right T-DNA border. The red rectangle indicates the region where heterologous sequence is cloned. P35S, 2x35S promoter from the
cauliflower mosaic virus. Splice donor and acceptor sequences flank the LIR within circularized replicons (data not shown). Blue and red arrows indicate
primer binding sites for PCR to detect circularized GVRs.
(B) Images of leaf tissue expressing GFP and GUS. As a negative control for GVR-mediated protein expression, leaf tissue was infiltrated with a single
strain of Agrobacterium containing the LSL T-DNA plasmid. Tissue delivered pLSLGFP was imaged 3 d after infiltration. Tissue-delivered pLSLGUS was
stained in a solution with X-Gluc 7 d after infiltration.
(C) PCR-based detection of circularized GVRs within plant cells. The PCR control (LSL T-DNA) used primers designed to amplify sequence within linear
LSL T-DNA.
Replicons for Plant Genome Engineering 153
observed a maximum copy number of ;6000 per single-copy
gene. This is consistent with previous reports describing that
mastreviruses replicate to very high copy number within plant
cells (Timmermans et al., 1992). Together, these data illustrate
that our GVRs replicate and express heterologous proteins in leaf
cells. Furthermore, these results show that peak replicon and
protein levels occur between 3 and 5 d after infiltration, which is
consistent with previous reports using BeYDV-based replicons
for protein expression (Zhang and Mason, 2006). In subsequent
genome engineering experiments, we chose to sample tissues
between 5 and 7 d after infiltration. We reasoned that sampling on
these days would allow sufficient time for the GVRs to reach
maximum copy number and for peak protein expression. Furthermore,
as DNA modifications are stable events, a targeted
change occurring at 3 d after infiltration, for example, should be
detectable 7 d after infiltration.
Expression of ZFNs with GVRs
To demonstrate targeted mutagenesis using GVRs, an LSL T-DNA
plasmid was modified to encode the Zif268:FokI ZFN (pLSLZ;
Figure 2A). Target sequence for this ZFN is present within a reporter
gene that is stably integrated in tobacco plants (Wright
et al., 2005). This reporter is a nonfunctional, translational fusion
between GUS and neomycin phosphotransferase (NPTII; Datla
et al., 1991). Leaf tissue from transgenic tobacco plants carrying
the gus:nptII reporter was syringe infiltrated with an Agrobacterium
strain containing pLSLZ (one side of a leaf) or coinfiltrated with
a mixture of Agrobacterium strains containing pLSLZ and pREP
(the other side of the leaf). Total DNA was extracted 7 d after infiltration,
replicational release was verified by PCR (Figure 2A), and
the Zif268 target sequence was analyzed for mutations introduced
by NHEJ. To this end, a 484-bp DNA fragment encompassing the
Zif268 target sequence was amplified by PCR. Amplicons were
digested with MseI (an MseI site is present in the spacer sequence
between the two Zif268 binding sites), and bands were resolved
by agarose gel electrophoresis (Figure 2B). Cleavage-resistant
amplicons from leaf tissue transformed with both pLSLZ and
pREP T-DNA were cloned and sequenced (Figure 2C). Six out of
eight sequenced clones contained mutations at the ZFN target
site. Five out of the six sequences had distinct NHEJ-induced
mutations, indicating that DNA in multiple leaf cells had been
modified. We observed significantly higher levels of targeted
mutagenesis in cells transformed with pLSLZ and pREP compared
with pLSLZ alone (Figure 2D). Cleavage-resistant amplicons
observed from tissue delivered pLSLZ T-DNA are most likely due
to expression of Zif268:FokI from the virion-sense LIR promoter.
These results indicate that GVRs can express ZFNs for creating
targeted DNA double-strand breaks.
Expression of TALENs and Components of the CRISPR/Cas
System with GVRs
Having demonstrated that GVRs can deliver ZFNs to plant cells,
we next tested if they were effective vehicles for delivering TALENs
and CRISPR/Cas reagents. We constructed a pLSL vector that
encodes the T30 TALEN pair (pLSLT; Supplemental Figure 8A;
Zhang et al., 2013), which targets a site within the tobacco
acetolactate synthase (ALS) genes. A second vector was constructed
that encodes Cas9 and synthetic guide RNA (sgRNA),
also targeting ALS (pLSLC; Supplemental Figure 8D). After delivery
to plant cells, mutagenesis of endogenous targets was monitored
by loss of a restriction enzyme site due to NHEJ-induced mutation
at the site of cleavage. We observed an enhancement in mutagenesis
when pREP was cotransformed, suggesting GVR-mediated
expression of TALENs and CRISPR/Cas elements can be achieved
(Supplemental Figures 8B, 8C, 8E, and 8F).
GVRs Enhance Frequencies of Gene Targeting
GVRs were next assessed for their ability to achieve gene targeting
through the delivery of sequence-specific nucleases and
repair templates. The target for modification was the defective
Figure 2. GVR-Mediated Expression of ZFNs for Targeted Mutagenesis.
(A) LSL T-DNA encoding Zif268:FokI (top). PCR-based detection of circularized
GVRs within plant cells (bottom).
(B) PCR-based detection of ZFN-induced mutations at the Zif268 target
sequence. Numbers beneath the gel image indicate the percentage of
cleavage-resistant amplicons. NTC, nontransformed control.
(C) Sequences of individual amplicons containing NHEJ-induced mutations.
Letters in front of gray background indicate the Zif268 binding
sequence.
(D) Quantification of NHEJ-induced mutations. Error bars represent SE of
three experiments. *P < 0.05.
154 The Plant Cell
gus:nptII gene, which can be repaired by correcting a 600-bp
deletion that removes part of the coding sequences for both GUS
and NPTII (Wright et al., 2005). An LSL T-DNA plasmid was constructed
that encodes the Zif268:FokI ZFN followed immediately
by repair template sequence (designated us:NPTII) designed to
restore gus:nptII function (pLSLZ.D; Figure 3A). Cells having undergone
gene targeting will express a functional GUS protein and,
consequently, will stain blue when incubated in a solution with the
GUS substrate X-Gluc. Random integration of the repair template
or read-through transcription from virus promoters should not
produce functional GUS protein due to 500 bp that is missing from
the 59 end of the coding sequence. This was experimentally
confirmed by transforming nontransgenic leaf tissue with pLSLZ.D
and pREP T-DNA; no GUS activity was observed 5 d after infiltration
(Figure 3B, Neg. Ctrl.). To compare the performance of
GVRs with conventional T-DNA, we engineered a T-DNA vector
with Zif268:FokI sequence followed immediately by the us:NPTII
repair template (p35SZ.D). Five days after infiltration, leaf tissue
was stained in X-Gluc and assessed for cells having undergone
gene targeting. We observed an enhancement in GUS activity in
leaf tissue transformed with pLSLZ.D and pREP T-DNA (Figure 3B)
relative to the p35SZ.D and pLSLZ.D controls. To verify repair of
the reporter gene, PCR was performed on total DNA extracted
from leaf tissue transformed with pLSLZ.D and pREP T-DNA.
Primers were used that recognize sequence within the repaired
reporter gene and ;1 kb downstream of the homology carried by
the repair template (Supplemental Figure 9). Amplification of
a 2.3-kb product from 9 of the 11 leaf samples indicates the
presence of the repaired GUS:NPTII transgene. Sequence analysis
confirmed the presence of an NPTII repair template-transgene
junction, consistent with repair of the reporter by gene targeting.
The relative enhancement in gene targeting was calculated by
quantifying the density of blue sectors within transformed leaf
tissue. A significant enhancement in gene targeting was observed
in tissue transformed with pLSLZ.D and pREP T-DNA across
multiple transgenic plant lines where the reporter gene is integrated
at different chromosomal positions (Figure 3C; Wright et al., 2005).
Notably, in plant line 1, we observed an enhancement in gene
targeting greater than two orders of magnitude. Taken together,
these results demonstrate that GVRs can deliver sequence-specific
nucleases and repair templates for gene targeting and that GVRs
contribute features that result in the enhancement of gene targeting
in somatic cells relative to conventional T-DNA delivery.
Double-Strand Breaks Enhance Gene Targeting
There are several features of GVRs that may promote gene targeting,
including replication to high copy numbers, high protein
expression and pleiotropic activities of Rep and RepA. To test
these features, we transformed single leaves with two Agrobacterium
samples and directly compared GUS activity resulting
from gene targeting (Figure 4A). Pairing two samples on a single
leaf reduced the variation in results due to environmental conditions
(e.g., differences in leaf age, size, and health). As T-DNA
size affects transfer efficiency, we ensured that vectors being
directly compared had similar T-DNA sizes. To determine the
contribution of double-strand breaks to gene targeting and to test
the effectiveness of this assay, Zif268:FokI (897 bp) was replaced
with GFP (912 bp). We observed a significant decrease (100-fold)
in GUS activity when Zif268:FokI was removed (Figure 4B), which
is consistent with previous reports describing a stimulatory effect
of double-strand breaks on recombination (Puchta et al., 1996).
Replication of Repair Templates Enhances Gene Targeting
To determine if replication of GVRs contributes to gene targeting,
we compared our GVR system (pLSLZ.D and pREP) to
a replication-inactive system (p35SZ.D and pREP). We observed
Figure 3. GVRs Promote High-Frequency Gene Targeting.
(A) Approach to repair a nonfunctional gus:nptII gene through homologous recombination. GT, gene targeting; TS, Zif268:FokI target site.
(B) Images of leaf tissue stained in a solution with X-Gluc. To better visualize stained cells, chlorophyll was removed from leaf tissue, and the green and
blue channels were removed from the image. The Neg. Ctrl. image is of nontransgenic leaf tissue transformed with two strains of Agrobacterium
containing pLSLZ.D and pREP.
(C) Relative frequencies of gene targeting using GVRs (pLSLZ.D + pREP; blue bars) and conventional T-DNA (p35SZ.D; red bars). The x axis represents
four different plant lines with the gus:nptII gene integrated at different chromosomal positions. Error bars represent SE of at least three biological
replicates. *P < 0.05 and **P < 0.005.
Replicons for Plant Genome Engineering 155
a significant decrease in blue sectors when the cis-acting replication
elements were removed (Figure 4C, left graph). These
results indicate that replication enhances gene targeting; however,
we could not conclude whether replicating sequencespecific
nucleases or repair templates contributed more toward
gene targeting. Therefore, to address this issue, we explored the
role of sequence-specific nuclease expression on GVR-mediated
gene targeting.
One explanation for the observed high frequency of gene targeting
is that GVRs mediate high expression of sequence-specific
nucleases, and, as a result, double-strand breaks are more
frequent. To test this hypothesis, we compared the level of NHEJinduced
mutations in leaf tissue delivered Zif268:FokI using GVRs
(pLSLZ and pREP) or conventional T-DNA (p35SZ). We predicted
that if expression of sequence-specific nuclease was enhanced
using GVRs, we would observe higher levels of NHEJ-induced
mutations. Our results refute this hypothesis (Supplemental
Figure 10A): We observed a lower level of NHEJ-induced mutations
with GVRs, suggesting that replication of sequence-specific
nucleases does not augment levels of gene targeting. As an
alternative approach to assess the impact of nuclease amplification,
we modified Rep and RepA to carry a mutation in the
conserved ATPase domain (K219H for BeYDV). This mutation
impairs replication (Desbiez et al., 1995; Supplemental Figure
10B); however, other functions of Rep and RepA should remain
active (e.g., nicking activity, ligation activity, and transactivation
of virion-sense transcription). As expected, pREPK219H T-DNA
delivered to leaf tissue was not replicated (Supplemental Figure
10C), and we observed similar levels of targeted mutagenesis
with pREPK219H as with pREP (;10%; Supplemental Figures
10D and 10E). Collectively, the results suggest that replication of
Zif268:FokI coding sequence does not significantly contribute to
enhancing targeted mutagenesis.
To test the hypothesis that gene targeting is enhanced by replication
of repair templates and not sequence-specific nucleases,
we compared gene targeting frequencies of two GVR systems that
either replicate sequence-specific nucleases or repair templates
(Figure 4C, right graph). Here, we observed significantly higher
levels of gene targeting when repair templates were replicated.
Similarly, when we translocated repair templates from conventional
Figure 4. Synergism between Double-Strand Breaks, Replication of Repair Templates, and Pleiotropic Activity of Rep and RepA on Gene Targeting.
(A) Illustration of the approach to directly compare gene targeting frequencies between two Agrobacterium samples.
(B) Effect of targeted double-strand breaks on gene targeting. As indicated, red arrows represent coding sequence for Zif268:FokI; black boxes depict
the us:NPTII repair templates (RT); small black boxes represent the left and right T-DNA borders. Error bars represent SE of six experiments. ***P < 0.001.
Data were normalized to sample 1.
(C) Effect of replicating ZFN and repair template sequence on gene targeting (left graph). Error bars represent SE of four experiments. ***P < 0.001. Effect
of replicating repair template sequence on gene targeting (right graph). Error bars represent SE of three experiments. **P < 0.005. Data were normalized
to sample 1 in both graphs.
(D) Effect of Rep and RepA expression on gene targeting. Measure of center is mean. Error bars represent SE of four experiments. *P < 0.05. Data were
normalized to sample 1.
156 The Plant Cell
T-DNA to GVRs, we observed substantially higher levels of gene
targeting (Supplemental Figure 11). These results indicate that although
expression of sequence-specific nucleases is critical for
gene targeting, replication of repair template sequence contributes
more toward enhancing gene targeting.
Pleiotropic Activity of Rep and RepA Enhances
Gene Targeting
Geminivirus Rep and RepA proteins interact with a multitude of
host proteins. Specifically, RepA protein from mastreviruses
interacts with Geminivirus RepA binding protein 1 (GRAB1) and
GRAB2 (Xie et al., 1999) and the retinoblastoma-related protein
(RBR; Horváth et al., 1998). It may be possible that expression of
mastrevirus Rep and RepA in plant cells affects levels of homologous
recombination. To test this hypothesis, we compared
gene targeting levels in tissue-delivered p35SZ.D T-DNA or
p35SZ.D and pREP T-DNA. Here, we observed a significant
increase in blue sectors when pREP was delivered (Figure 4D).
To confirm that delivery of pREP T-DNA to plant cells results in
the expression of both Rep and RepA, we used RT-PCR to
detect Rep and RepA transcripts. To avoid detecting RepA
transcripts produced by Agrobacterium, we transfected protoplasts
with the pREP T-DNA plasmid. Total RNA was extracted
24 h after transfection, and we detected unspliced (RepA) and
spliced (Rep) transcripts (Supplemental Figure 12). These results
demonstrate that pleiotropic activity of Rep and/or RepA promotes
gene targeting (Figure 4D). Taken together with the
results in the previous two sections, high-frequency gene targeting
using GVRs is due to synergy between targeted double-strand
breaks, replication of repair template sequence, and pleiotropic
activity of Rep and RepA.
Recovery of Calli and Plantlets with a Precise DNA Change
To demonstrate the feasibility of GVRs for generating fully developed
plants with a desired sequence change, we regenerated
cells harboring the corrected GUS:NPTII transgene. We chose to
regenerate leaf cells because our previous data demonstrate the
usefulness of GVRs in whole leaves. Leaves were syringe infiltrated
with two strains of Agrobacterium containing pLSLZ.D
and pREP. Five days later, infiltrated leaves were removed from
the plant, surface sterilized, and placed on regeneration media
with kanamycin (Figure 5A). To track the progression of regenerating
cells with the GUS:NPTII transgene, subsets of leaf
discs were stained in X-Gluc solution 0, 7, 14, 21, 42, and 49 d
after plating. We observed GUS expression from leaf discs
stained between 0 and 21 d (Figures 5A to 5D). After day 42, we
observed shoots with GUS expression (Figures 5E and 5F). To
confirm that tissue staining blue harbors the corrected GUS:NPTII
transgene, we extracted genomic DNA from blue calli and performed
PCR with primers designed to amplify sequence from the
repaired GUS:NPTII transgene. Because our negative control
samples (noninfiltrated tissue) did not produce calli or shoots, we
chose to analyze genomic DNA from callus tissue that did not
stain blue in the pLSLZ.D and pREP samples. We observed
amplification of sequence from all four blue-stained tissues and
not from tissue that did not stain blue (Figure 5G). Sequencing of
all four PCR products confirmed the presence of an NPTII repair
template-transgene junction, consistent with repair of the reporter
by gene targeting (Figure 5H). These findings demonstrate that
GVRs can be used to introduce exact DNA changes in plant cells
and that these cells maintain the ability to regenerate.
Development of a Single-Component GVR System
Having demonstrated the usefulness of GVRs for genome engineering
and that there are virus-specific features that promote
gene targeting, we sought to improve the design of the geminivirus
vectors for efficient genome editing. To this end, we developed
a single-component system that requires the delivery of only one
T-DNA molecule to generate GVRs. Transfer of a single T-DNA to
cells is more efficient than the transfer of two T-DNAs, which may
result in a higher frequency of cells receiving a functional GVR. We
modified the GVR sequence such that Rep and RepA coding sequences
were placed downstream of the complementary-sense
LIR promoter, the natural position of Rep and RepA coding sequences
within mastrevirus genomes. Furthermore, we positioned
the strong constitutive promoter (P35S) directly upstream of our
reporter gene or sequence-specific nuclease of interest (Figure 6A).
To test the effectiveness of this vector system for protein
expression, we placed GFP coding sequence downstream of
P35S (pLSLGFP.R; Supplemental Figure 13A). We compared
protein expression of pLSLGFP.R to the dual-component GVR
system (pLSLGFP and pREP). In general, we observed more
uniform GFP expression with pLSLGFP.R (Supplemental Figure
13B). This is most likely due to a higher number of cells receiving
a functional GVR and from transient expression of GFP from the
linear LSL T-DNA. To verify that replicational release had occurred,
total genomic DNA was extracted from leaf tissue expressing
GFP and PCR was performed using primers that were
homologous to sequences within GFP and the Rep/RepA coding
sequences. Strong amplification of LIR sequence indicated
the presence of circular GVRs (Supplemental Figure 6).
To assess gene targeting with our single-component vector
system, we placed Zif268:FokI and us:NPTII repair template sequence
downstream of the 2x35S promoter and within the LIR
borders (pLSLZ.D.R; Figure 6A; Supplemental Figure 14). One
side of a leaf was syringe infiltrated with Agrobacterium containing
pLSLZ.D.R and the other with was infiltrated with a mixture
of Agrobacterium containing pLSLZ.D and pREP. As an
additional control, a separate leaf was infiltrated with p35SZ.D.
Five days after infiltration, leaf tissue was stained in a solution
containing X-Gluc (Figure 6B) and the density of blue spots was
quantified (Figure 6C). We observed comparable levels of GUS
activity using our single-component vector relative to the twocomponent
system. In general, we noticed that tissue delivered
pLSLZ.D.R had more uniform GUS activity. This observation may
reflect a difference in the transfer efficiency of a single T-DNA
molecule to that of two T-DNA molecules.
Having previously demonstrated that gene targeting is stimulated
by replication of repair templates, and not sequencespecific
nucleases, we predicted that positioning Zif268:FokI
coding sequence outside of the LIR borders (but still within the
same T-DNA; pZLSLD.R; Figure 6A) would result in comparable
gene targeting frequencies to pLSLZ.D and pREP. Our results
Replicons for Plant Genome Engineering 157
support this hypothesis. Gene targeting frequencies of pZLSLD.
R were similar to that of the two-component system (pLSLZ.D
and pREP; Figure 6C). Together, these results illustrate the
usefulness of single-component vectors for the delivery of GVRs
for recombination-based genome editing.
DISCUSSION
The potential of geminiviruses for gene targeting has long been
recognized, principally because their DNA genomes could serve
as repair templates, and they replicate to high copy number. We
found the key to realizing this potential was the use of sequence-specific
nucleases to stimulate recombination between
the virus and the chromosomal target. In addition, replication of
repair templates and pleiotropic activities of Rep and RepA
further enhanced gene targeting; however, these factors were
insufficient to achieve gene targeting in the absence of a targeted
chromosome break. Together, these data stress the importance
of using geminivirus technology in conjunction with
sequence-specific nucleases.
Genome size constraints hindered the use of full viruses for
genome engineering. One strategy to increase the cargo capacity
of begomoviruses is to replace the coat protein gene with a desired
heterologous sequence of up to 800 bp. This strategy
permits the delivery of some genome engineering tools, including
repair templates, meganucleases, ZFN monomers, and possibly
sgRNA for the CRISPR/Cas system. However, the cargo capacity
is insufficient for expression of large heterologous sequences,
Figure 5. Regeneration of Cells with the Repaired GUS:NPTII Transgene.
(A) Image of a leaf disc stained in X-Gluc 0 d after plating.
(B) Image of a leaf disc stained in X-Gluc 7 d after plating.
(C) Image of leaf discs stained in X-Gluc 14 d after plating.
(D) Image of a leaf disc stained in X-Gluc 21 d after plating.
(E) Shoots with GUS activity 42 d after plating.
(F) Shoots with GUS activity 49 d after plating.
(G) PCR-based detection of the repaired GUS:NPTII transgene. Total DNA was extracted from four calli that stained blue. As a negative control,
genomic DNA was extracted from callus that did not stain blue (Callus #5). Primers were designed to be homologous to sequence within the 600-bp
modification sequence and ;1 kb downstream of the homologous DNA region carried by the repair template. The genomic DNA control used primers
designed to amplify sequence within the endogenous F-box gene.
(H) DNA sequences of amplicons generated from calli #1 to #4.
158 The Plant Cell
including TALENs and Cas9. For large cargo, there is a high
likelihood that larger than wild-type genomes will recombine to
smaller sizes. Virus genome size constraints are imposed by cellto-cell
movement through plasmodesmata and not by replication
(Gilbertson et al., 2003). Therefore, to deliver large heterologous
sequences, we deleted virus genes involved in cell-to-cell movement
and retained the elements required for rolling-circle replication.
This effectively eliminated constraints on cargo capacity
and made it possible to simultaneously deliver sequence-specific
nucleases and repair templates for gene targeting. In addition, this
approach allowed us to maintain the ability for replicons to amplify
to high-copy number by rolling-circle replication.
Compared with meganucleases and ZFNs, TALENs and CRISPR/
Cas reagents are, in general, easier to construct and have increased
efficacy. Furthermore, TALENs appear to have low frequencies of
off-target cleavage (Mussolino et al., 2011), and CRISPR/Cas
technology enables efficient multiplexed genome engineering (Cong
et al., 2013; Wang et al., 2013). Therefore, it would be advantageous
for new vectors for genome engineering to be compatible with
TALEN and CRISPR/Cas technology. Our results indicate that GVRs
accomplish this. By placing TALENs and Cas9/sgRNAs within
GVRs, we achieved targeted mutagenesis of the endogenous ALS
genes. For gene targeting, when the repair template is on the GVR,
sequence-specific nucleases can be expressed using either GVRs
or conventional T-DNA, both of which resulted in comparable levels
of gene targeting. Taken together, these results suggest that not
only are GVRs amenable for expression of TALENs and the
CRISPR/Cas system, but they can be combined with other methods
for delivery of sequence-specific nucleases, such as T-DNA.
We observed that gene targeting was enhanced by pleiotropic
activity of Rep and RepA. One hypothesis to explain these results
derives from our understanding of how geminiviruses establish
successful infection within host plants. Geminiviruses only infect
nondividing cells and encode proteins that promote entry into
S-phase (Horns and Jeske, 1991; Lucy et al., 1996; Kong et al.,
2000; Ascencio-Ibáñez et al., 2008). Progression into S-phase is
thought to provide geminiviruses with necessary host factors
required for replication (e.g., nucleotides and DNA polymerase). In
monocots, S-phase entry is mediated by the mastrevirus RepA
protein: RepA interacts with the plant’s RBR and sequesters RBR
from E2F, thereby promoting cell cycle progression into S-phase.
The BeYDV RepA has been shown to interact with RBR from
maize (Zea mays), and our results suggest that RepA stimulates
tobacco cells to enter S-phase (Liu et al., 1999). The basis for this
latter conclusion is that in higher-eukaryotic cells, the frequency
of homologous recombination increases as cells move out of
a resting state and into S and G2 (Rothkamm et al., 2003). We
believe promotion into S-phase by Rep/RepA underlies the
stimulatory effect these proteins have in gene targeting.
Geminiviruses infect a wide range of plants, including both monocots
and dicots. We modified the BeYDV, which belongs to the
genus Mastrevirus. Most mastreviruses infect monocots; however,
the BeYDV infects dicots, including tobacco species, Datura stramonium,
tomato (Solanum lycopersicum), French bean (Phaseolus
vulgaris), and Arabidopsis (Liu et al., 1997). Whereas BeYDV replicons
should function within this list of species, they are unlikely to
replicate in plants that are not hosts to the wild-type virus, including
monocots. It may be possible to develop GVR systems for monocots
using other mastreviruses (e.g., wheat dwarf virus and maize streak
virus). Both wheat dwarf virus and maize streak virus have long been
used for protein expression in monocots and replicate to similar copy
numbers as our BeYDV replicon system (Timmermans et al., 1992;
Palmer et al., 1999). Thus, there is promise for GVRs to enable efficient
genome engineering across many plant species.
METHODS
Vector Construction
LSL T-DNA plasmids (pLSL, pLSLGUS, pLSLGFP, pLSLZ, pLSLT, pLSLC,
and pLSLZ.D) were constructed using the Agrobacterium tumefaciens
binary vector pCAMBIA1300. LIR and SIR sequences were derived from the
mild BeYDV strain (Halley-Stott et al., 2007) (GenBank accession number
DQ458791). The boundaries of the LIR and SIR sequences were defined
following previously described methods (Mor et al., 2003). The cauliflower
mosaic virus promoter (P35S) was positioned within the replicon and
Figure 6. Single-Component Geminivirus Vectors Enable Efficient Genome
Editing.
(A) Illustration of single-component GVR vectors. The blue box represents
SIR sequence.
(B) Selected images of leaves transformed with pLSLZ.D and pREP,
pLSLZ.D.R, pZLSLD.R, or p35SZ.D. Green and blue channels were removed
from the images to better visualize GUS staining.
(C) Quantification of GUS activity in leaf tissue delivered GVR constructs.
The left graph represents data from three leaves transformed with
pLSLZ.D and pREP on one side and pLSLZ.D.R on the other side. The
right graph represents data from two leaves transformed with pLSLZ.D
and pREP on one side and pZLSLD.R on the other. Error bars represent
SE. P values for both graphs were >0.05.
Replicons for Plant Genome Engineering 159
upstream of the LIR. To enable translation of coding sequence that is
located downstream of the LIR, splice donor and acceptor sequences were
positioned upstream and downstream, respectively, of the LIR sequence
that is formed upon circularization. To enable facile cloning, pLSL was
modified to harbor Gateway-compatible attR1 and attR2 sequences between
the upstream LIR and SIR elements. Conventional T-DNA expression
plasmids (pREP, pREPK219H, p35SZ, and p35SZ.D) were constructed
using the Gateway-compatible Agrobacterium binary vector pMDC32.
Coding sequence for Rep and RepA was derived from the mild BeYDV
strain (Halley-Stott et al., 2007).
Two entry vectors were constructed for cloning sequence-specific nuclease
(or reporter protein) and repair template sequences into the pLSL and
pMDC32 destination plasmids. The first entry vector (pNJB091) is a derivative
of pZHY013 (Zhang et al., 2013) with attL2 sequence replaced with Nos-T and
attL5 sequence. The second entry vector (pNJB080) is also a derivative of
pZHY013, but here the FokI and attL1 sequences were replaced with the
chloramphenicol resistance gene, ccdB gene, and attL5 sequence.
To engineer pLSL vectors for the repair of the gus:nptII transgene,
pNJB80 was modified to harbor repair template sequence (us:NPTII), and
pNJB91 was modified to harbor the Zif268:FokI ZFN coding sequence. To
generate the us:NPTII sequence for cloning into pNJB80, pDW1269 (Wright
et al., 2005) was used as a template in a PCR with the oligonucleotides
59-CGCGGAGCGGCCGCAGCAGTCTTACTTCCATGATTTC-39 and 59-GATCGATCTCTGCAGCCCCGCGCGTTGGCCGATTCA-39.
Bold letters indicate
NotI and PstI recognition sites for cloning into pNJB80. The resulting repair
template contained 600 bp of corrective sequence flanked by 1 kb of homologous
sequence to the gus:nptII reporter. To generate Zif268:FokI sequence
for cloning into pNJB91, pDW1345 (Wright et al., 2005) was used as
a template in a PCR with the oligonucleotides 59-GCCCTTCACCATGGCTTCCTCCCCTCCAAAGAAAAAG-39
and 59-GAACGATCGGACGTCCTATTAAAAGTTTATCTCACCGTTA-39.
Bold letters indicate the NcoI and AatII
restriction enzyme sites used for cloning into pNJB91. The resulting plasmids
were used in a MultiSite Gateway (Life Technologies) recombination reaction
with pLSL or pMDC32 to generate pLSLZ.D or p35SZ.D, respectively. pLSLZ
and p35SZ were generated as described above; however, instead of using
pNJB80 with the us:nptII sequence, we used a different pNJB80 vector which
harbors ;3.0 kb of filler DNA sequence.
To generate pLSLT, pNJB91 was modified to harbor the T30 TALEN pair
sequence (Zhang et al., 2013). This sequence contains the two T30 TALEN
open reading frames linked by a T2A translational skipping sequence. The
N- and C-terminal truncation for both TALENs was ND152/C63 (Miller et al.,
2011). The T30 TALEN sequence was cloned from pZHY528 into pNJB91
using standard cloning techniques. The resulting plasmid was used in
a MultiSite Gateway recombination reaction with pLSL and pNJB80
containing the us:NPTII repair template to generate pLSLT.
To generate pLSLC, pNJB91 was modified to harbor a codon-optimized
Cas9 gene from Streptococcus pyogenes, and pNJB80 was modified to
harbor the Arabidopsis thaliana U6 RNA Pol III promoter followed by sgRNA
sequence. The Cas9 gene was codon optimized for dicotyledonous plants
and synthesized (GenScript) with C- and N-terminal SV40 NLS signals and
an N-terminal 3x FLAG-tag sequence. The U6:gRNA sequence was
synthesized on gBlocks (Integrated DNA Technologies).
Single-component LSL vectors (pLSLGFP.R, pLSLZ.D.R, and pZLSLD.R),
where Rep/RepA coding sequence was positioned within the replicon, were
constructed in T-DNA plasmids with the pCAMBIA backbone. Sequence of
features within pLSLZ.D.R can be found in Supplemental Figure 14.
Boundaries for the LIR and SIR were determined using the annotated sequence
for the mild BeYDV strain (GenBank accession number DQ458791).
Plant Material
Tobacco (Nicotiana tabacum var Xanthi) plants, both wild type and
transgenic (Wright et al., 2005), were grown at 21°C with 60% humidity
under a 16-h-light and 8-h-dark cycle. Leaves (fully expanded upper
leaves) from 4- to 6-week-old tobacco plants were used for Agrobacterium
transformation. When comparing gene targeting or targeted
mutagenesis frequencies between different T-DNA molecules, one leaf
per plant was infiltrated.
Agrobacterium Transformation
T-DNA vectors were introduced into Agrobacterium (GV3101/pMP90) using
the freeze-thaw method. Single transformed colonies were grown overnight
at 28°C in 3 mL of Luria-Bertani starter culture containing kanamycin (50 mg/mL)
and gentamycin (50 mg/mL). The next day, 1 mL of starter culture was
used to inoculate 50 mL of Luria-Bertani culture containing kanamycin
(50 mg/mL), gentamicin (50 mg/mL), 10 mM MES, and 20 mM acetosyringone.
After reaching an OD600 of ;1 (around 16 h), cells were pelleted and
resuspended to an OD600 of 0.2 using infiltration buffer (10 mM MES, 150 mM
acetosyringone, and 10 mM MgCl2, pH 5.6). Resuspended cultures were
incubated at room temperature for 2 to 4 h. For experiments requiring the
coinfiltration of two different Agrobacterium strains, cultures were mixed
together in a 1:1 ratio, or, for three different strains, in a 1:1:1 ratio. When
comparing two different Agrobacterium samples, the ODs of the solutions
were adjusted accordingly to ensure equal concentrations of
individual Agrobacterium strains. Leaves from tobacco plants were
infiltrated with Agrobacterium using a 1-mL syringe. Immediately following
infiltration, plants were watered and covered with a plastic dome
to maintain high humidity. Plastic domes were removed ;24 h after
infiltration.
GUS Assay
To visualize cells expressing GUS, leaf tissue was vacuum infiltrated with
X-Gluc solution (10 mM phosphate buffer, 10 mM EDTA, 1 mM ferricyanide,
1 mM ferrocyanide, 0.1% Triton X, and 1 mM X-Gluc). Leaf tissue
was then incubated in X-Gluc solution at 37°C for ;24 h. Chlorophyll was
removed by incubating stained leaf tissue in 80% ethanol for ;1 week.
Tobacco Regeneration
Leaves from 4- to 5-week-old tobacco plants were infiltrated with pLSLZ.D
and pREP. As a control for selection and regeneration of kanamycin resistant
cells, additional leaves were infiltrated with pCAMBIA2301 (T-DNA
that contains a kanamycin resistance and also the GUS gene). Two
separate experiments were performed; for each experiment, three to five
leaves from different plants were infiltrated with each T-DNA or T-DNA pair.
Five days after infiltration, leafs were removed from the plant and surface
sterilized by submersion in 10% bleach for ;20 min. Leaves were then
washed three times in sterile water. Sterilized leaf tissue was cut into ;1 3
1-cm discs using a surgical blade. Leaf discs were plated onto regeneration
media (4.4 g Murashige and Skoog medium with vitamins, 1 mg/L
6-benzylaminopurine, 0.1 mg/L 1-naphthaleneacetic acid, 30 g Suc, and 8 g
agar, pH 5.7) containing kanamycin (100 mg/mL) and Timentin (200 mg/mL).
As a negative control for tobacco selection, nontransformed leaf tissue was
sterilized and plated. Discs were transferred to new plates weekly.
PCR-Based Detection of Circularized GVRs and the GUS:NPTII
Transgene
To detect the presence of circularized GVRs, total DNA was extracted
from plant tissue and used as a template for PCR. Oligonucleotides were
designed to be homologous to DNA sequence located upstream and
downstream of LIR sequence in the circular replicon (59-GTTTCACTTCACACATTATTACTG-39
and 59-TGTTGAGAACTCTCGACGTCCTGC-
39). To minimize strand transfers during amplification, PCR was performed
using the Expand Long Template PCR system (Roche).
160 The Plant Cell
To detect the presence of a repaired GUS:NPTII transgene, total DNA
was extracted from leaf or callus tissue and used as a template for PCR.
Oligonucleotides were designed to be homologous to DNA sequence
located within the corrective sequence on the repair template and sequence
;1 kb downstream of the region of homology harbored on the
repair template (59-GTCGGTGAACAGGTATGGAAT-39 and 59-CTACA-
TACCTCGCTCTGCTAATC-39).
Detection of ZFN-, TALEN-, and Cas9-Induced Mutations
Experiments that used GVRs to mediated expression of ZFNs, TALENs,
and CRISPR/Cas elements for targeted mutagenesis were performed
similarly: One side of a leaf was delivered pLSL, and the other side was
infiltrated with pLSL and pREP. Five to seven days after infiltration, total
DNA was extracted and assessed for mutations at the appropriate
sequence-specific nuclease target site. To detect Zif268:FokI-induced
mutations, the gus:nptII target locus was amplified using primers that are
homologous to sequences 270 bp upstream and 214 bp downstream of
the MseI site (present within the spacer region of the Zif268 binding
sequences; 59-AAGGTGCACGGGAATATTTCGCGC-39 and 59-GCCATGATGGATACTTTCTCG-39).
The resulting amplicons were digested for 16
h at 37°C with MseI and resolved by gel electrophoresis. Cleavageresistant
amplicons were gel purified, cloned into pJET1.2, and sequenced.
To detect TALEN-induced mutations at the SurA and SurB loci, total DNA
was first predigested with AluI for 16 h at 37°C. Digested DNA was used as
a template for a PCR with primers designed to amplify a 573-bp product
encompassing the T30 binding site (59-GACGGTGCAGAAAGTGAAGTA-
39 and 59- TATGGCCCAGGAGTGTCTAA-39). The resulting amplicons
were digested for 16 h at 37°C with AluI and resolved by gel electrophoresis.
Cleavage-resistant amplicons were gel purified, cloned into
pJET1.2, and sequenced. To detect Cas9-induced mutations, the target
DNA sequence present within SurA and SurB was amplified by PCR
(forward, 59-TGACATCTGGTGGATTAGGAGC-39; reverse, 59-CAATCACATCCAACAAGTATGGC-39).
The resulting 408-bp amplicons were
digested with AlwI for 16 h at 37°C. Cleavage-resistant products
were gel purified and cloned into pJET1.2 and sequenced.
Data Collection and Statistics
Following staining in X-Gluc, the density of blue sectors was quantified in
leaf tissue. Three nonoverlapping images were taken for each infiltrated
region (leaf halves) using a camera attached to a stereoscope (Nikon).
Each image captured 1.8 cm2 of leaf tissue, totaling 5.4 cm2 for each
leaf half. Image locations were directed to capture the entire number
of blue sectors for each leaf half. For samples where the total number
of blue sectors exceeded 5.4 cm2 of area, regions with the highest
density of blue sectors were imaged. Leaves without detectable GUS
activity were excluded from the data analysis. ImageJ software was
used to calculate the number of blue sectors per image. P values in
Figures 4 to 6 were generated using a paired, two-tailed Student’s t
test. P values in Figures 2 and 3 were generated using a two-tailed
Student’s t test.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: ADH1, AT1G77120.1; mild strain of BeYDV, DQ458791; SurA,
X07644.1,SurB,X07645.1;pCPCbLCVA.007,AY279345;andpCPCbLCVB.002,
AY279344.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Engineering Cabbage Leaf Curl Virus for
Delivery of Repair Templates in Arabidopsis.
Supplemental Figure 2. Phenotype of Arabidopsis Plants Infected
with Cabbage Leaf Curl Virus.
Supplemental Figure 3. Temporal Control of Targeted Double-Strand
Breaks in Whole Plants.
Supplemental Figure 4. PCR-Based Detection of Geminivirus-Mediated
Gene Targeting.
Supplemental Figure 5. Time Course of Geminivirus RepliconMediated
Expression of Green Fluorescent Protein.
Supplemental Figure 6. PCR-Based Detection of Circularized Geminivirus
Replicons.
Supplemental Figure 7. Time Course of Geminivirus Replicon Gene
Copy Number.
Supplemental Figure 8. Expression of TALENs and Components of
the CRISPR/Cas System Using Geminivirus Replicons.
Supplemental Figure 9. PCR-Based Detection of the Repaired GUS:
NPTII Transgene in Tobacco Leaf Cells.
Supplemental Figure 10. Replication of Sequence-Specific Nucleases
Does Not Enhance Targeted Mutagenesis.
Supplemental Figure 11. Exploring the Role of Repair Templates for
Geminivirus Replicon-Mediated Gene Targeting.
Supplemental Figure 12. Detection of Rep and RepA Transcripts in
Tobacco Protoplasts.
Supplemental Figure 13. Expression of Green Fluorescent Protein
with Single-Component Geminivirus Replicon Vectors.
Supplemental Figure 14. Sequence of Features within the SingleComponent
Geminivirus Vector pLSLZ.D.R.
Supplemental Methods.
ACKNOWLEDGMENTS
We thank members of the Voytas lab for the helpful discussion and
insights with this work. We thank Dominique Robertson for the kind
donation of the CaLCuV plasmids. This research was supported by
a grant to D.F.V. from the National Science Foundation (DBI 0923827)
and by a grant to J.G.-H. from Fundación Alfonso Martín Escudero.
AUTHOR CONTRIBUTIONS
N.J.B. and D.F.V. designed the research. N.J.B., J.G.-H., and T.C.
performed the experiments. N.J.B. analyzed the data. N.J.B., T.C., and
P.A.A. contributed reagents/materials/analysis tools. N.J.B. and D.F.V.
wrote the article.
Received October 21, 2013; revised December 8, 2013; accepted
December 18, 2013; published January 17, 2014.
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Replicons for Plant Genome Engineering 163
DOI 10.1105/tpc.113.119792
; originally published online January 17, 2014;2014;26;151-163Plant Cell
Nicholas J. Baltes, Javier Gil-Humanes, Tomas Cermak, Paul A. Atkins and Daniel F. Voytas
DNA Replicons for Plant Genome Engineering
This information is current as of October 23, 2014
Supplemental Data
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http://www.plantcell.org/content/suppl/2013/12/23/tpc.113.119792.DC1.html
References
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