Letters
https://doi.org/10.1038/s41477-019-0386-z
An efficient DNA- and selectable-marker-free
genome-editing system using zygotes in rice
Erika Toda 1,2*, Narumi Koiso2, Arika Takebayashi1, Masako Ichikawa3, Takatoshi Kiba 1,
Keishi Osakabe4, Yuriko Osakabe1,4, Hitoshi Sakakibara 1, Norio Kato1,2,3 and Takashi Okamoto
Technology involving the targeted mutagenesis of plants
using programmable nucleases has been developing rapidly
and has enormous potential in next-generation plant breed-
ing. Notably, the clustered regularly interspaced short pal-
indromic repeats (CRISPR)–CRISPR-associated protein-9
nuclease (Cas9) (CRISPR–Cas9) system has paved the way
for the development of rapid and cost-effective procedures
to create new mutant populations in plants1,2. Although
genome-edited plants from multiple species have been pro-
duced successfully using a method in which a Cas9–guide
RNA (gRNA) expression cassette and selectable marker are
integrated into the genomic DNA by Agrobacterium tumefa-
ciens-mediated transformation or particle bombardment3,
CRISPR–Cas9 integration increases the chance of off-target
modifications4, and foreign DNA sequences cause legislative
concerns about genetically modified organisms5. Therefore,
DNA-free genome editing has been developed, involving the
delivery of preassembled Cas9–gRNA ribonucleoproteins
(RNPs) into protoplasts derived from somatic tissues by poly-
ethylene glycol–calcium (PEG–Ca2+)-mediated transfection
in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine,
apple8 and potato9, or into embryo cells by biolistic bombard-
ment in maize10 and wheat11. However, the isolation and cul-
ture of protoplasts is not feasible in most plant species and
the frequency of obtaining genome-edited plants through
biolistic bombardment is relatively low. Here, we report a
genome-editing system via direct delivery of Cas9–gRNA
RNPs into plant zygotes. Cas9–gRNA RNPs were transfected
into rice zygotes produced by in vitro fertilization of isolated
gametes12 and the zygotes were cultured into mature plants in
the absence of selection agents, resulting in the regeneration
of rice plants with targeted mutations in around 14–64% of
plants. This efficient plant-genome-editing system has enor-
mous potential for the improvement of rice as well as other
important crop species.
In animals, to produce genetically heritable traits of interest, in
vitro transcribed Cas9 messenger RNA (mRNA) and single-guide
RNA (sgRNA) or preassembled Cas9 protein–sgRNA complexes
are delivered into zygotes (one-celled embryos) by direct injec-
tion, resulting in the production of bi-allelic mutant animals with
high efficiency13,14. By contrast, such a direct delivery system into
zygotes has not been established for angiosperms, since fertiliza-
tion of angiosperms occurs in embryo sacs deeply embedded in
ovaries15,16. Recently, we developed a polyethylene glycol–calcium
(PEG–Ca2+)-mediated transfection system for egg cells isolated
Plant Breeding Innovation Laboratory, RIKEN Cluster for Science, Technology and Innovation Hub, Yokohama, Japan. 2Department of Biological Sciences,
1
Tokyo Metropolitan University, Hachioji, Japan. 3Plant Innovation Center, Japan Tobacco Inc., Iwata, Japan. 4Faculty of Bioscience and Bioindustry,
Tokushima University, Tokushima, Japan. *e-mail: toda-erika1@ed.tmu.ac.jp; okamoto-takashi@tmu.ac.jp
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1,2
*
from rice flowers17, making it possible to deliver macromolecules
such as DNA into isolated gametes. Our previous study demon-
strated that plasmid DNA was not delivered into rice zygotes that
had been mechanically isolated from pollinated rice ovaries17. This
result can be explained by the fact that the cell walls that surround
the plasma membranes impede the access of macromolecules to
these membranes18–21. Isolated rice egg cells do not possess an evi-
dent cell wall12; formation of cell walls around the plasma mem-
branes of zygotes generally starts after gamete fusion, and cell walls
are thought to be immature in the early zygotes12,22,23. Therefore,
in the present study, we prepared rice zygotes by electro-fusion of
isolated egg and sperm cells, and subjected them to PEG–Ca2+-
mediated transfection with plasmid within 1 h after gamete fusion
(Supplementary Fig. 1a). To monitor the delivery of the plasmid
into rice zygotes and subsequent transient gene expression, we used
pGFP-ER, a plasmid encoding green fluorescent protein (GFP)
tagged with signal peptide (SP) and the carboxy-terminal His-Asp-
Glu-Leu (HDEL) sequence under control of the 35S promoter and
HSP70 intron, for PEG–Ca2+-mediated transfection. The SP–GFP–
HDEL protein is detected efficiently in plant cells, including rice
egg cells17. The fluorescent signal was detected 0.8 d after fusion and
continued to be visible in globular-like embryos and cell masses for
4 d after fusion (Supplementary Fig. 1b). This indicated that the
delivered DNAs were transcribed, and the resulting transcripts were
translated in the zygotes before the first cell division. The intensity
of the signal decreased 8 d after fusion, suggesting that the deliv-
ered plasmid was degraded at the cell-mass stage around 8 d after
PEG–Ca2+-mediated transfection. The cell mass developed into a
white callus and regenerated into a plantlet (Supplementary Fig. 1b
and Table 1). Of 14 zygotes PEG–Ca2+-transfected with pGFP-ER,
10 exhibited GFP-derived fluorescent signals. This indicates an effi-
ciency of PEG–Ca2+-mediated transfection of in vitro-produced rice
zygotes with pGFP-ER of 71.4%, which is higher than the approxi-
mately 30% achieved with rice egg cells17.
Transgenic rice plants expressing DsRed2 were prepared and the
zygotes produced by the fusion of gametes from these transformants
were subjected to further analyses using the Cas9–gRNA system.
These transgenic plants were used because targeting of gRNA to the
DsRed2 sequence in the zygotes is assumed to induce target muta-
genesis in the DsRed2 sequence and lead to a decrease in DsRed2
expression in the zygotes and/or subsequent embryos. When
zygotes produced by fusion of gametes from the transgenic plants
were cultured, the level of DsRed2-derived fluorescent signals grad-
ually increased until 8 d after fusion (Fig. 1a), and the cell masses
regenerated into plantlets (results not shown). RNPs consisting
Letters NATuRE PlAnTS

Table 1 | Developmental profiles of PEG–Ca2+-transfected zygotes and efficiency of the targeted mutation in regenerated plantlets
Cross Transfected materials No. of No. of zygotes that developed to each growth stage Frequency
PEG–Ca2+- of targeted
Plasmid Expressed Expressed Two- Globular Cell White Plantlet
transfected mutation (%)
DNA or or delivered or delivered celled embryo-like mass callus (plant
zygotes
RNP protein gRNA embryo structure lines)
Egg (WT) × DNA SP–GFP–HDEL – 14 9 8 6 5 3 –
sperm (WT)
Egg RNP Cas9 gDsRed2 12 11 11 11 9 7 3/12 (25)
(DsRed2)
× sperm
(DsRed2)
Egg (WT) × RNP Cas9 gDL-2 21 19 18 18 18 15 3/21 (14.3)
sperm (WT) RNP Cas9 gDL-3 22 22 20 20 20 16 3/22 (13.6)
RNP Cas9 gGW7 14 13 13 13 13 12 3/14 (21.4)
RNP Cas9 gGCS1 14 14 14 14 13 12 9/14 (64.3)
DNA Cas9 gDL-2 12 10 10 9 9 7 2/12 (16.7)
DNA Cas9 gPRR37-1 25 23 23 22 20 17 4/25 (16)
DNA Cas9 gPRR37-2 1/25 (4)
WT, wild type. RNP, ribonucleoprotein.

of Cas9 protein and sgRNA targeted to the DsRed2 sequence were
assembled and delivered into rice zygotes expressing DsRed2
via PEG–Ca2+-mediated transfection (Fig. 1b). Of 12 transfected
zygotes, 7 developed and regenerated into plantlets (Table 1). When
DsRed2-derived signals were monitored during the development of
these seven zygotes, we detected different fluorescent patterns, no
signal, a strong signal, or a weak signal in the cell masses at 6–13 d
after fusion (Fig. 1c). The disappearance of the DsRed2 signal was
detected in cell masses derived from five zygotes (zygotes R1, R3, R4,
R5 and R7, see Supplementary Table 1). In these zygotes, the DsRed2
signals gradually decreased following 1 d after fusion (Fig. 1d).
Verification of the targeted mutations in the plant lines derived
from the five zygotes (no. 1, 3, 4, 5 and 7) resulted in identification
of homozygotic mutations in two plant lines (no. 1 and no. 5; Fig. 1e,
Supplementary Fig. 2 and Supplementary Table 1). However, for
the remaining three plant lines (no. 3, 4 and 7), PCR amplification
of the target region in the DsRed2 sequence was unsuccessful. This
could be explained by a large deletion of genomic DNA around the
DsRed2 target site, although we have no direct evidence for this. In
the target sequence of plant line no. 2, in which the DsRed2 signal at
the cell mass stage was weaker than that in the non-treated cell mass
(Fig. 1c, right), a mono-allelic deletion was detected (Fig. 1e). Thus,
among 12 treated zygotes, 3 plant lines with targeted mutations were
obtained, indicating a mutation frequency of 25% (Table 1).
For targeted mutagenesis of endogenous genes, wild-type rice
zygotes produced by gamete fusion were used. The DROOPING
LEAF (DL) gene was selected as one target, since knockout pheno-
types of DL and gRNA sequences for this gene (gDL-2 and gDL-3)
have been reported previously24. Twenty-one zygotes were PEG–
Ca2+-transfected with RNPs consisting of Cas9 protein and the
sgRNA of gDL-2, and 15 plant lines were regenerated via zygote
culture (Table 1). Among these 15 plant lines, three plant lines
(no. 1, 14 and 15) had targeted mutations (Fig. 2a, Supplementary
Figs. 3 and 4a, and Supplementary Table 2). Similarly, 22 zygotes
were PEG–Ca2+-transfected with RNPs consisting of Cas9 protein
and the sgRNA of gDL-3, another target sequence of DL, and 16
plant lines were regenerated (Table 1). A homozygotic mutation was
detected in plant line no. 4 (Fig. 2b,c, Supplementary Figs. 4b and 5,
and Supplementary Table 3), and the mutant line showed a droop-
ing leaf phenotype (Fig. 2d). In addition, mono-allelic mutations
were detected in two plant lines, no. 13 and no. 14. The efficiency
of targeted mutagenesis of the DL gene via RNP transfection was
about 14% (Table 1). RNP-mediated genome editing was further
conducted for the promoter sequence of the GRAIN WIDTH 7
(GW7) gene, and for the exon sequence of the GENERATIVE CELL
SPECIFIC-1 (GCS1) gene. From 14 zygotes PEG–Ca2+-transfected
with RNPs containing gRNA targeted to GW7 or GCS1, 3 and 9
plant lines were obtained with targeted mutations in GW7 and
GCS1, respectively (Fig. 2e,f, Table 1, Supplementary Figs. 6–8, and
Supplementary Tables 4 and 5).
In addition to RNP-mediated target mutagenesis of endogenous
genes, we also performed genome editing of the DL gene using a plas-
mid vector. Twelve zygotes were PEG–Ca2+-transfected with a plas-
mid containing a Cas9-gRNA (gDL-2) expression cassette (Fig. 3a),
resulting in seven plant lines (Table 1). Among these plant lines,
targeted mutations were detected in two plant lines, no. 1 and no.
4 (Fig. 3b, Supplementary Fig. 9 and Supplementary Table 6). Next,
dual genome editing was tested for the promoter sequence of the
Pseudo-Response Regulator 37 (PRR37) gene. The transfer RNA-
based-multiplex CRISPR–Cas9 vector containing two types of
gRNAs for the PRR37 sequence (gPRR37-1 and gPRR37-2) was pre-
pared (Fig. 3c) and used for PEG–Ca2+-mediated transfection. After
culturing 25 treated zygotes, 17 plant lines were obtained (Table 1).
Among these 17 plant lines, mutations in the sequences targeted
by gPRR37-1 and gPRR37-2 were detected in four plant lines (no.
5, 6, 14 and 17) and one plant line (no. 6), respectively (Fig. 3d,
Supplementary Fig. 10 and Supplementary Table 7). Among the
four lines with the gPRR37-1 mutation, one regenerated plant, no.
6-1, harboured a double mutation, indicating that multiple targeted
mutagenesis is possible. However, the remaining three lines (no. 5,
14 and 17) had mutations in the PRR37-1 site alone. The difference
in the targeting efficiency between these two gRNAs was most likely
due to the secondary structure of the guide sequence25. Prediction of
the secondary structures of the gRNAs for PRR37-1 and PRR37-2
indicated that a stem-loop structure is formed through complemen-
tary binding in the target-sequence region in gPRR37-2, but not in
gPRR37-1 (Supplementary Fig. 11).
For the genome-edited plants produced by PEG–Ca2+-
transfection of plasmids with the DL and PRR37 genes, the possible
integration of the plasmid DNA into genomic DNA was examined
using genomic PCR for the DNA sequence of the Cas9 protein and
hygromycin resistance gene on the plasmid vector. Integration was

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NATuRE PlAnTS Letters
a detected in one (no. 1) of two dl plant lines and one (no. 17) of four
prr37 plant lines (Fig. 3e). These data indicate that RNP-mediated
genome editing can be used to avoid the integration of foreign DNA
sequences that can occur with plasmids, which is a cause of legisla-
tive concerns about genetically modified organisms5.
Genome-edited animals have been widely and efficiently pro-
duced by the delivery of CRISPR–Cas9 components into zygotes via
direct injection; this is because the zygote, a totipotent cell develop-
0 1 4 6 8 10 ing into embryo, is the most suitable target for delivering CRISPR–
Days after fusion Cas9 components. Here, we have established methods for genome
b Electro-fusion RNP
editing in rice by direct delivery of CRISPR–Cas9 RNPs or vectors
Zygotic
PEG–Ca2+ development
into zygotes produced by electro-fusion of isolated gametes with a
transfection and high frequency of targeted mutations of around 4–64% (Fig. 3f and
Sperm induction of Table 1). Therefore, the present study represents a milestone in the
targeted gene
cell Egg cell Transfected modification
production of genome-edited plants by direct delivery of CRISPR–
Zygote zygote Cas9 components into plant zygotes possessing totipotency.
c In this study, rice zygotes produced by electro-fusion of isolated
gametes were used as target cells for delivery of CRISPR–Cas9 com-
ponents. Although the applicability of the methods, gamete isolation
and electro-fusion, is not wide and requires some technical skills,
maize and wheat zygotes can be also produced by electro-fusion26,27,
suggesting that application of the methods to maize and wheat is
achievable. Moreover, utilization of zygotes produced by electro-
– ++ +
fusion will not be essential, since PEG–Ca2+-mediated transfection
of maize zygotes isolated from pollinated flowers, treated with cell
d
wall-degrading enzymes, has been reported17. Importantly, the pro-
cedure for the isolation of zygotes from pollinated flowers is much
simpler than that of electro-fusion of gametes. In addition, the pro-
cedures for zygote isolation have been established in several major
crops, and isolated maize, wheat and barley zygotes can be cultured
into regenerated plants28–30. These suggest that technical limitations
in the present genome-editing approach with plant zygotes pro-
0 1 4 6 8 11 duced by electro-fusion will soon be overcome by the use of zygotes
Days after fusion isolated from cereal flowers.
e DsRed2 Rice, maize and wheat provide the majority of the world’s human
no. 1 –1 food energy intake, and innovative solutions are needed to improve
no. 2 –36 these crops to produce enough food for the growing human popu-
0 lation. Implementation of the methods reported here, involving a
no. 5 –4 DNA- and selectable-marker-free genome-editing system using
plant zygotes, has the potential to advance the molecular breeding
Fig. 1 | Genome editing in rice by direct delivery of CRISPR–Cas9 of improved types of these important cereals.
RNP into zygotes. a, Developmental profiles of a rice zygote
expressing DsRed2. The zygote produced by gamete fusion using
rice transformants expressing DsRed2 (0 d) was observed with a Methods
Plant materials. Oryza sativa L. cv. Nipponbare and cv. Yukihikari were grown
fluorescent microscope 1 (one-celled stage), 4, 6, 8 and 10 d after fusion in an environmental chamber (Nippon Medical and Chemical Instruments) at
(cell mass stages). b, A schematic illustrating the procedure used to 26 °C/24 °C with a 13 h light/11 h dark photoperiod. Preparation of transformed
produce zygotes by electro-fusion of rice gametes and subsequent rice plants expressing DsRed2 is described in Supplementary Methods.
PEG–Ca2+-mediated transfection of these zygotes with RNPs. Pink,
Isolation and electro-fusion of gametes. The isolation of egg and sperm cells from
green and orange circles indicate the egg, sperm and zygotic nuclei,
rice flowers was performed as described31, and electro-fusion of isolated egg and
respectively. The grey circles and yellow flash indicate RNPs and the sperm cells for zygote production was conducted as reported12. For isolation of egg
point of electro-fusion, respectively. c, Zygotes were produced as in cells, ovaries collected from rice flowers before flowering were transferred to plastic
Fig. 1a, PEG–Ca2+-transfected with CRISPR–Cas9 RNPs containing the dishes (35 mm diameter) containing 3 ml mannitol solution (370 mOsmol kg−1
DsRed2 target sequence, and cultured for 6 or 13 d. DsRed2 fluorescent H2O) and cut transversely with a razor blade. From the lower parts of the dissected
ovary, an egg cell was released. Using a glass capillary connected to a handling
profiles in cell masses were observed, and typical images showing
injector, the isolated egg cells were transferred into a 0.5–1.0 µl mannitol droplet
no DsRed2 signal (left), a strong DsRed2 signal (middle) and a weak in mineral oil on a coverslip under an inverted microscope31 (IX-73, Olympus).
DsRed2 signal (right) are presented. d, A rice zygote expressing DsRed2 To isolate sperm cells, anthers collected from rice flowers before flowering were
was produced and PEG–Ca2+-transfected as in Fig. 1b. The treated transferred to mannitol solution (370 mOsmol kg−1 H2O) in plastic dishes (35 mm
zygote (0 d) was cultured and observed 1 (one-celled stage), 4, 6, 8 diameter). The isolated anthers were torn to release the pollen grain using forceps.
After 3–5 min, the pollen grains burst due to osmotic shock and released their
and 11 d after fusion (cell mass stages). e, Mutant DNA sequences at contents, including sperm cells31.
the DsRed2 target in plant lines that were regenerated from the PEG– For electro-fusion of gametes, approximately eight to ten mannitol
Ca2+-transfected zygotes. The target sequence in DsRed2 is underlined. droplets (fusion droplets) including an egg cell and a sperm cell were prepared.
The protospacer-adjacent motif (PAM) sequence is shown in red. Top Thereafter, the electro-fusion apparatus (ECFG21, Nepa Gene) and electrodes
and bottom panels in a,c and d represent fluorescent and bright-field (CUY5100Ti100, Nepa Gene) were assembled, and the position of the electrodes
in a fusion droplet was adjusted. After alignment of an egg cell and a sperm cell on
images, respectively. Scale bars: 20 µm (a, day 0, 1 and 4; d, day 0, 1 and one of the electrodes under an alternating current field (1 MHz, 3–5 V root mean
4), 50 µm (a, day 6 and 8; c, left and middle; d, day 6 and 8), 100 µm square), approximately 0.5 µl of the mannitol solution (520 mOsmol kg−1 H2O) was
(a, day 10) and 200 µm (c, right; d, day 11). gently added to the fusion droplet using a glass capillary. Thereafter, gamete fusion

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Letters NATuRE PlAnTS

a DL (gDL-2)
WT
no. 1–1 –50
0
no. 14 –8
0
no. 15–1 +1 (A) c
+63, –11 WT
no. 15–2 +1 (A)

b DL (gDL-3)
40 130 120
WT
no. 4 +1 bp (C)
no. 4 +1 (C)
*
no. 13–2 –52, +16
0
no. 14 +1 (C)
0
130 120 110

d f
WT gDL-3 no. 4 GCS1
WT
no. 3–1 –14
–30
no. 3–2 –1, +5
–30
no. 4–1 –14
–30
no. 4–2 –14, +4
0
no. 5 –18, +12
no. 6–1 –2
DL : +/+ –/– 0
no. 7–1 –9
e 0
GW7 no. 8 –16, +3
WT –3
no. 7 +2 (AA) no. 9 –1, +56
no. 10–1 +1 (A) no. 11 +1 (T)
0 0
no. 10–2 +1 (A) no. 12 +1 (T)
no. 12 +1 (A) –42
0

Fig. 2 | Targeted mutagenesis of endogenous genes in rice by delivery of the CRISPR–Cas9 RNPs into zygotes. a,b, Mutant DNA sequences at the DL
locus in plant lines derived from zygotes PEG–Ca2+-transfected with CRISPR–Cas9 RNPs, consisting of Cas9 protein and gRNA for DL, gDL-2 (a) or gDL-3
(b). WT, wild type. c, Sanger sequencing chromatograms at target sites of gDL-3 in a wild-type plant, and in plant line no. 4, which was genome-edited
by the CRISPR–Cas9 RNPs. The asterisk and underlined text indicate the inserted nucleotide and PAM sequences, respectively. d, The phenotype of the
homozygotic mutant plant line no. 4, shown in b and c, displaying the drooping leaf phenotype. e,f, Mutant DNA sequences at the promoter sequence
of GW7 (e) and the exon sequence of the GCS1 locus (f) in plant lines derived from zygotes PEG–Ca2+-transfected with RNPs. In a,b,e and f, the inserted
nucleotides and PAM sequences are shown in light blue and red, respectively, and the target sequences are underlined.

was induced by a single negative direct-current pulse (50 µsec, 11–15 V), resulting
in the production of the fused cell (zygote)12. The produced zygotes were subjected
to PEG–Ca2+-mediated transfection. More detailed protocols for electro-fusion of
gametes and subsequent culture of rice zygotes are described in ref. 32.
PEG–Ca2+-mediated transfection of zygotes with CRISPR–Cas9–gRNA RNPs
and subsequent culture of zygotes. A Cas9-nuclease protein tagged with a
nuclear localization signal (GeneArt Platinum Cas9 Nuclease or TrueCut Cas9
Protein v2) was purchased from Thermo Fisher Scientific. sgRNA was synthesized
and purified using the GeneArt Precision gRNA Synthesis Kit (Thermo Fisher
Scientific). All primers used for in vitro transcription of sgRNA are listed in
Supplementary Table 8. For genome editing using an RNP, Cas9 protein (1 µg)
and in vitro transcribed sgRNA (2–3 µg) were premixed in Cas9 protein storage
buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.6 mM TCEP and 50% glycerol)
in a total volume of 1.5–2.1 µl and incubated at room temperature for 10 min.
After incubation, the solution containing the RNP complexes was gently mixed
with 10 µl of MMG solution (4 mM MES-KOH, pH 5.7, 15 mM MgCl2 in mannitol
solution of 450 mOsmol kg−1 H2O) (MMG-RNP), and the MMG-RNP was used for
the PEG–Ca2+-mediated transfection of zygotes. PEG–Ca2+-mediated transfection
using zygotes was conducted as described in ref. 17 with minor modifications.
Three to six rice zygotes were transferred into a 0.5–1.0 µl droplet of mannitol
solution (450 mOsmol kg–1 H2O) overlaid with mineral oil on a coverslip, and
then washed twice by transfer into droplets of MMG solution. The zygotes were
transferred into a 1.0 µl droplet of MMG-RNP, and then a 1.0 µl droplet of PEG
solution (30% PEG4000, Sigma-Aldrich, and 100 mM CaCl2 in mannitol solution
in 450 mOsmol kg−1 H2O) was merged with the droplet of MMG-RNP including
the zygotes. Immediately after merging, the resulting solution was mixed gently
with a glass capillary for 1 min. After incubation for 5 min, approximately half to
an equal amount of mannitol solution (450 mOsmol kg−1 H2O) was added into the
mixed droplet. Thereafter, approximately half the volume of the solution in the
mixed droplet was aspirated using a glass capillary connected to a manual injector.
To reduce the PEG concentration, an equal volume of mannitol solution was
delivered into the droplet, and, after mixing the droplet gently, approximately half
the solution was removed from the droplet. This procedure was repeated five to six
times. Thereafter, the zygotes were washed three or four times by transfer into fresh
droplets of mannitol solution. Subsequently, the PEG–Ca2+-transfected zygotes
were cultured in a Millicell-CM insert (Merck Millipore) with N6Z medium as
described12, and the cell masses from the cultured zygotes were regenerated into
plantlets as described33.
Multiplex CRISPR–Cas9 vector construction. The construction of the tRNA-
based multiplex CRISPR–Cas9 vector that was utilized for both single and
multiplex gRNA expression is described in detail in Supplementary Methods. In
brief, a vector harbouring an O. sativa codon-optimized Streptococcus pyogenes
Cas9 (OsCas9) sequence, fused to GFP via a self-cleaving 2A peptide under the
control of the ubiquitin promoter, and the tRNA–gRNA units under aOsU3
gene promoter, was used (Supplementary Tables 9 and 10). The target
sequences for the tRNA–gRNA units were selected using the focas webtool34

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NATuRE PlAnTS Letters
(http://focas.ayanel.com). To construct the tRNA–gRNA units for single genome
a pT 3 × NLS Ter
engineering, the target sequence was amplified from an artificial gene including
RB OsU3 tRNA gRNA Ubi coCas9 GFP Hyg LB tRNA and gRNA scaffold sequences using specific primers (Supplementary
2A Table 8). To construct the tRNA–gRNA units for multiplex genome engineering,
tRNA–gRNA1–tRNA–gRNA2 was amplified from an artificial gene including
tRNA and gRNA scaffold sequences using specific primers (Supplementary
b DL (gDL-2) Table 8). The amplified target sequence or tRNA–gRNA fragment was inserted
WT into the BsaI site of multiplex CRISPR–Cas9 vectors using Golden Gate Cloning
no. 1 –1/+1 methods (New England Biolabs).
no. 4 +1
+1
PEG–Ca2+-mediated transfection of zygotes with plasmid vectors. Using the
c multiplex CRISPR–Cas9 vector described above, MMG solution containing
pT 3 × NLS Ter plasmid (MMG-DNA, 80–320 ng µl−1) was prepared. PEG–Ca2+-mediated
RB OsU3 tRNA gRNA1 tRNA gRNA2 Ubi coCas9 GFP Hyg LB transfection of zygotes with plasmid DNA was conducted as above except for the
2A
use of MMG-DNA instead of MMG-RNP.

Microscopy. The fluorescence of GFP in cells was observed under an IX-73

inverted fluorescent microscope (Olympus) with 460–495 nm excitation and 510
d PRR37 (gPRR37-1) (gPRR37-2) nm emission wavelengths (U-FBW mirror unit, Olympus). The fluorescence of
WT WT DsRed2 in cells was observed with 530–550 nm excitation and 575 nm emission
no. 5 +1 WT wavelengths (U-FGW mirror unit, Olympus). Digital images of zygotes and their
no. 6–1 –4 –13
no. 6–2 –4 WT
resulting embryos were obtained using a cooled CCD camera (DP73, Olympus)
no. 14 +1 WT and cellSens software (Olympus).
–4 WT
no. 17 +1 WT Genomic DNA PCR and sequencing of target sites. Genomic DNAs were
prepared from leaves of rice plants, regenerated from zygotes that had been
id

e
T)

PEG–Ca2+-transfected with CRISPR–Cas9 components, using PrepMan Ultra

T)

sm

prr37 rice plants

d

(W

dl rice plants
(W
mi

pla

(Thermo Fisher Scientific) according to the manufacturer’s instruction. PCR

las

nt
nt

1
1

2
pla
37

–1

–2
–1
–2
2p

pla

4–

7–
4–

7–
–1

–2
–2

–1

fragments containing the target site were amplified using PrimeSTAR GXL DNA
RR

.5

.6
.6
.5

.1
ce

.1
.1

.1
.1
L-

.4
.1

.4
ce

no

no
no
no
gP

no
gD

no
no

no
Ri
no

no
no

no
Ri

polymerase (Takara) with the primer set for each target (Supplementary Table 8).
Cas9 The amplified PCR fragments were purified by ExoSAP-IT Express PCR Cleanup
Reagents (Thermo Fisher Scientific), and the purified PCR fragments were
Hygromycin
sequenced using an ABI 3130 Genetic Analyzer. For samples with heterologous
target mutations, the amplified PCR fragments were further subcloned into
Control
the pCR-Blunt vector using the Zero Blunt PCR Cloning Kit (Thermo Fisher
Scientific), and plasmids from four or more independent clones of transformed
CRISPR-Cas9 vector Isolation of gametes
preparation In vitro fertilization Culture in Plantlet Genomic PCR Escherichia coli were sequenced.
or sgRNA synthesis 2+
PEG-Ca transfection liquid medium regeneration sequencing Genomic DNA PCR for examining integration of plasmid DNA into genomic
f DNA was conducted with primer sets (Supplementary Table 8) for the CRISPR–
6 or 2 1 18–20 30–45 3–4 Duration
(days) Cas9 vector that was used for PEG–Ca2+ transfection.

RNP or
Induction of targeted
gene modification
Electro-fusion vector
PEG–Ca2+
transfection
Sperm
cell Egg cell
Zygote Transfected Two-celled Cell mass
zygote embryo
Fig. 3 | Genome editing in rice by direct delivery of the tRNA-based
CRISPR–Cas9 multiplex vector into zygotes. a, Construction of a tRNA-
based CRISPR–Cas9 vector for genome editing of the targeted sequence
in DL (gDL-2). OsU3, O. sativa U3 snRNA gene promoter; Ubi, ubiquitin
promoter; coCas9, Cas9-coding sequence; Hyg, hygromycin resistance
gene. NLS, nuclear localization signal; pT, poly T; 2A, self-cleaving 2A
peptide; Ter, OsHSP16.9A gene terminator. b, Mutant DNA sequences at
the target site of gDL-2 in the plant lines derived from zygotes PEG–Ca2+-
transfected with the CRISPR–Cas9 vector. c, Construction of a tRNA-
based multiplex CRISPR–Cas9 vector for genome editing of the promoter
sequence in PRR37. The vector harbours dual target sites, gPRR37-1 and
gPRR37-2. d, Mutant DNA sequences at the target sites of gPRR37-1 and
gPRR37-2 in the plant lines derived from PEG–Ca2+-transfected zygotes
with the CRISPR–Cas9 vector. e, Integration of plasmid DNA into genomic
DNAs in the dl and the prr37 rice plant lines. Genomic DNAs isolated from
the regenerated rice plants with target mutations shown in b and d were
subjected to PCR for the DNA sequence of the Cas9-coding sequence
and the hygromycin resistance gene on the vector. The GCS1 gene on the
genomic DNA was used as a PCR control. f, Flow chart of the genome-
editing process in rice by direct delivery of the CRISPR–Cas9 RNP or vector
in zygotes. The duration in days of each procedure is shown. Pink, green
and orange circles indicate the egg, sperm and zygotic nuclei, respectively.
The yellow flash and green leaf shapes indicate the point of electro-fusion
and plant leaves, respectively. In b and d, the inserted nucleotides and PAM
sequences are shown in light blue and red, respectively, and the target
sequences are underlined.
Prediction of the secondary structures of guide RNAs. The secondary structures
Regen- of the gRNAs were predicted using RNA Folding Form software35, available at the
eration
mfold web server (http://unafold.rna.albany.edu/?q=mfold/rna-folding-form).
Genome-edited Statistics and reproducibility. For Fig. 1a, five zygotes expressing DsRed2
plantlet
were produced and their developmental profiles were independently observed
with similar results. For Fig. 1c,d, the experiment was repeated three times
independently and similar results were obtained. For Fig. 2c, sequencing was
repeated two times independently with similar results. For Fig. 2d, the observation
was repeated two times using independent plant lines and similar phenotypes
were obtained. For Fig. 3e, the experiment was repeated one to three times
independently with similar results.
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.
Data availability
Information on the multiplex vector (pMgPoef4_129-2A-GFP) has been deposited
in GenBank with the accession number LC460477. The authors declare that all
other data supporting the findings of this study are available in the paper and
its supplementary information files or from the corresponding author upon
reasonable request.
Received: 5 August 2018; Accepted: 13 February 2019;
Published: xx xx xxxx
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Acknowledgements
We thank A. Ide (RIKEN Cluster for Science) for performing part of the molecular
experiments, the RIKEN Bio Resource Center (Tsukuba) for providing cultured rice
cells (Oc line), and the RIKEN CSRS Sequencing Service (Yokohama) for sequencing.
This work was supported, in part, by the MEXT KAKENHI (Grant-in-Aid for Scientific
Research on Innovative Areas, grant no. 17H05845 to T.O.) and JSPS KAKENHI
(Grant-in-Aid for Challenging Exploratory Research, grant no. 16K14742 to T.O.). This
work was supported by JST’s Program on Open Innovation Platform with Enterprises,
Research Institute and Academia (OPERA) (Y.O.).
Author contributions
E.T., T.K., N.Kato and T.O. designed the experiments; E.T. performed most of the
experiments; N.Koiso provided technical assistance to E.T. and performed some parts of
the experiments; A.T. performed some parts of the molecular experiments; M.I. prepared
transformed rice plants; T.K. provided technical assistance; K.O. and Y.O. performed
vector construction; H.S., N.Kato and T.O. supervised the project; and E.T. and T.O.
conceived the project and wrote the article.
Competing interests
T.O., N.Kato, E.T., N.Koiso, M.I. and T.K. are co-inventors on a patent application
covering the results described in this paper.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41477-019-0386-z.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to E.T. or T.O.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature Limited 2019
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Data collection Digital images were obtained using a cooled CCD camera and cellSens Standard software version 1.15 (Olympus).

Data analysis The target sequences for the tRNA-gRNA units were selected using the web-tool "focas" version 0.40 (http://focas.ayanel.com). The
secondary structures of the gRNAs were predicted using the RNA Folding Form software, version 2.3 energies (The mfold Web Server,
http://unafold.rna.albany.edu/?q=mfold/rna-folding-form). Targeted mutagenesis of the genome-edited plants was confirmed by Parallel
Editor in GENETYX software (GENETYX, version 19). Sanger sequencing chromatograms at target sites of genes on genomic DNA were
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