Arabidopsis - Purdue University...Supplementary information, Figure S1 (A) Schematic diagram of the...

Supplementary information, Figure S1 (A) Schematic diagram of the sgRNA and hSpCas9

expression cassettes in a single binary vector designed for Agrobacterium-mediated stable

transformation of Arabidopsis and rice.

The expression cassette of hSpCas9 was driven by the 35S promoter, whereas the sgRNA was

driven by the AtU6-26 promoter for Arabidopsis and OsU6-2 for rice. (B) The list of locus IDs

of target genes in Arabidopsis and rice. (C) Schematic of the Cas9/sgRNA-targeting sites for

BRI1, GAI, JAZ1, ROC5, SPP and YSA. (D) Representative T1 transgenic plants of the GAI

sgRNA1 target. The left one is a T1 plant with relatively normal vegetative growth, while the

right one is a plant with similar growth phenotype as gai dwarf mutants. They were screened

on MS plates for 5 days and transplanted in soil for 4 weeks before photographing. The bar

equals to 1 cm.

Supplementary information, Figure S2 The target sites designed for generation of stable

transgenic plants in Arabidopsis and rice.

The restriction sites for RFLP analysis of transgenic plants are in boxes. The PAM sequences

are highlighted in magenta and the target sequences in cyan.

Supplementary information, Figure S3 Targeted indel mutations induced by engineered

sgRNA:Cas9 at the BRI1 gene sgRNA1 site in Arabidopsis.

Alleles shown were amplified from genomic DNA isolated from 12 independent T1 transgenic

plants separately and sequenced after cloned into vectors. The wild type sequence is shown at

the top with the PAM sequence highlighted in magenta and the target sequence in cyan. Red

dashes, deleted bases; red bases, insertions or mutations. The net change in length is to the right

of each sequence (+, insertion; D, deletion). Note that some alterations have both sequence

insertions and deletions.







of each sequence (+, insertion; D, deletion). The number of clones representing each mutant

allele is shown in brackets.


sgRNA:Cas9 at the GAI gene sgRNA1 site in Arabidopsis.








sgRNA:Cas9 at the JAZ1 gene sgRNA1 site in Arabidopsis.








sgRNA:Cas9 at the ROC5 gene sgRNA1 site in rice.








sgRNA:Cas9 at the SPP gene sgRNA1 site in rice.





of each sequence (+, insertion). The number of clones representing each mutant allele is shown

in brackets.


sgRNA:Cas9 at the YSA gene sgRNA1 site in rice.






allele is shown in brackets. The plants #5 and #7 correspond to the two plants showing albino

leaf phenotype.


sgRNA:Cas9 at the YSA gene sgRNA2 site in rice.






allele is shown in brackets. The plants #5 and #6 correspond to the two plants showing albino

leaf phenotype.

Supplementary information, Table S1 Mutation detected in the T1 transgenic plants of Arabidopsis

Plant IDNo. of clones

sequenced

No. of clones with

mutant alleles detected

No. of different


1 9 7 4

2 8 8 8

3 8 3 3

4 10 8 7

5 7 7 4

6 8 5 4

BRI1 sgRNA1 7 7 6 5

8 7 4 4

9 10 8 5

10 10 9 6

11 6 4 4

12 8 6 6

Total 98 75 60

1 23 13 3

BRI1 sgRNA2 2 24 11 4

3 24 4 2

Total 71 28 9

1 10 6 4

2 9 7 2

BRI1 sgRNA3 3 6 4 3

4 9 5 2

Total 34 22 11

1 15 8 1

GAI sgRNA1 2 19 4 2

3 19 5 1

Total 53 17 4

1 16 1 1

2 18 5 3

JAZ1 sgRNA1 3 22 4 2

4 17 12 11

5 16 11 8

Total 89 33 25

Supplementary information, Table S2 Mutation detected in the T0 transgenic plants of rice

Plant IDNo. of clones

sequenced

No. of clones with


No. of different


1 33 27 1

2 27 19 5

3 31 29 1

ROC5 sgRNA1 4 41 28 2

5 33 33 2

Total 165 136 11

SPP sgRNA1 1 36 31 1

Total 36 31 1

1 17 10 2

2 10 5 1

3 13 8 1

4 8 5 3

5 15 15 2

6 8 3 1

YSA sgRNA1 7 20 20 1

8 10 5 1

9 14 10 2

10 8 3 1

11 17 12 1

12 17 14 2

Total 157 110 18

1 11 8 8

2 11 11 5

YSA sgRNA2 3 10 5 4

4 9 3 3

5 6 6 3

6 12 11 7

Total 59 44 30

Suppplementary information, Table S3 List of primers used in this study.

Usage Primer name Sequence（5‘-->3’）

Cloning YF-FP 1F ACACGCTCGAGATGGTGAGCAAGGGCGAGG

YF-FP 1R ACACGGTCGACACTAGTGGATCCGTGGCGGATCTTGAAGTTCAC

YF-FP 2F ACACGGGATCCACTAGTGTCGACGACCACATGAAGCAGCACGAC

YF-FP 2R ACACGGAGCTCTTACTTGTACAGCTCGTC

AtU6-26F AAGCTTCGTTGAACAACGGA

AtU6-26R CGAAGGGACAATCACTACTTCG

OsU6-F GGATCATGAACCAACGGCCT

OsU6-R AACACAAGCGACAGCGCG

Cas9-F TTACTCGAGATGGACTATAAGGACCACGACG

Cas9-R ATTGGATCCTTACTTTTTCTTTTTTGCCTGGC

sgRNA fusion AtU6-26-85F TTATTTTAACTTGCTATTTCTAGCTCTAAAACAGGTCTTCTC

GAAGACCCAATCACTACTTCGACTCTAGCTGTA

AtU6-26-85R GTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC

ACCGAGTCGGTGCTTTTTTTGTCCCTTCGAAGGGCCTTT

TPCR-OsU6F GCCAGTGTGCTGGAATTGCCCTTGGATCATGAACCAACGGCC

TPCR-OsU6R GCTCTAAAACAGGTCTTCTCGAAGACCCACACAAGCGACAGCGCG

Target oligo YF-FP sgRNA1 F GATTGTGAACTTCAAGATCCGCCA

YF-FP sgRNA1 R AAACTGGCGGATCTTGAAGTTCAC

BRI1 sgRNA1 F GATTGTGGGTCATAACGATATCTC

BRI1 sgRNA1 R AAACGAGATATCGTTATGACCCAC

BRI1 sgRNA2 F GATTGGACATACATGAGCTCCTGA

BRI1 sgRNA2 R AAACTCAGGAGCTCATGTATGTCC

BRI1 sgRNA3 F GATTGTAAGAGCTGACATAGCCTG

BRI1 sgRNA3 R AAACCAGGCTATGTCAGCTCTTAC

GAI sgRNA1 F GATTGATGAGCTTCTAGCTGTTCT

GAI sgRNA1 R AAACAGAACAGCTAGAAGCTCATC

JAZ1 sgRNA1 F GATTGGCAATAGGAAGTTCTGTCAA

JAZ1 sgRNA1 R AAACTTGACAGAACTTCCTATTGCC

ROC5 sgRNA1 F GTGTGCGGAGAACGACAGCCGGTC

ROC5 sgRNA1 R AAACGACCGGCTGTCGTTCTCCGC

SPP sgRNA1 F GTGTG GCGATTGGGAGCCTCGGCG

SPP sgRNA1 R AAACCGCCGAGGCTCCCAATCGCC

YSA sgRNA1 F GTGTGCGCGCCACCTCGGCCGAAG

YSA sgRNA1 R AAACCTTCGGCCGAGGTGGCGCGC

YSA sgRNA2 F GTGTGCGCGACGAGCACCTTCATG

YSA sgRNA2 R AAACCATGAAGGTGCTCGTCGCGC

RFLP PCR BRI1 1F GAATCTCTGACGAATCTATCC

BRI1 1R CACTCTTTCTTCATCCCATC

BRI1 2F GATGGGATGAAGAAAGAGTG

BRI1 2R CTCATCTCTCTACCAACAAG

GAI F TGTTATTAGAAGTGGTAGTGGAGTG

GAI R AGCCGTCGCTGTAGTGGTT

JAZ1 F CAACCATGAGTTTATTCCCTTGT

JAZ1 R GGATTTAGACAGGCGACAATAAC

ROC5 F CTTTGGGGGCCTCTTTGAC

ROC5 R ATCTGCGTGCGGCGATTC

SPP F GTGAGCCCGAAGAGGAGT

SPP R TCCCAATAAACCACACGCAC

YSA F ACTTCCTCCCTCTCCCTGT

YSA R CGCCTTCATCAGTGTGTTG

Supplementary information, Data S1 MATERIALS AND METHODS

Growth of Arabidopsis and rice plants

Arabidopsis thaliana ecotype Columbia (Col-0) was used in all experiments. Seeds were

sown on MS plates and stratified for 3 days at 4°C, then grown under long-day conditions (16

h light/8 h dark) at 22°C for 5 days before being transplanted in soil. Rice plants were grown

under standard greenhouse condition (16h light at 30°C /8h night at 22°C).

Vector construction

The coding sequence of hSpCas9 was cloned from vector pX2601 using primers Cas9-F and

Cas9-R (Table S2) and subcloned into pA7-GFP with XhoI and BamHI to replace the GFP

gene, which provided a 2x 35S promoter and a Nos terminator. Then the Cas9 expression

cassette was subcloned into the pBluescript SK+ vector (Stratagene Inc., San Diego, CA) and

designated 35S-Cas9-SK.

The AtU6-26 promoter was cloned from Arabidopsis wild type Col-0 genomic DNA by PCR

with primers AtU6-26F and AtU6-26R (Table S2) adding KpnI and XhoI on the two ends,

respectively, and put into the pEasy Blunt vector (Transgen Biotech, China). They were then

subcloned into the pBluescript SK+ vector (Stratagene Inc., San Diego, CA) using KpnI and

XhoI sites. The 85bp chimeric guide RNA region containing two BbsI digest sites was

amplified from the vector pX3301 by PCR using AtU6-26-85F and AtU6-26-85R (Table S2)

and fused to the AtU6-26 promoter, which resulted in AtU6-26SK. After the designed oligos

(20bp targeting sequences) were cloned into the BbsI sites, these chimeric RNA expression

cassettes between KpnI and SalI were either cloned into the 35S-Cas9-SK for transient assay,

or into the KpnI and EcoRI region of pCambia1300 vector (Cambia, Canberra, Australia)

together with the SalI and EcoRI fragment of the Cas9 expression cassette for stable

transformation of Arabidopsis.

The OsU6-2 promoter was cloned from rice wild type Nipponbare genomic DNA by PCR

using OsU6-F and OsU6-R (Table S2) and put into the pEasy Blunt vector (Transgen Biotech,

China). Then transfer PCR was conducted using TPCR-OsU6F and TPCR-OsU6R (Table S2)

to replace the AtU6-26 promoter in the AtU6-26SK vector, which produced the OsU6SK

vector with the 85nt guide RNA region. After target oligos were successfully inserted into the

BbsI sites of the OsU6SK vector, the chimeric RNA expression cassettes between KpnI and

HindIII were similarly cloned into the pCambia1300 vector (Cambia, Canberra, Australia)

between the KpnI and EcoRI sites together with the HindIII and EcoRI Cas9 expression

cassette for stable transformation of rice.

Transient YFP-HR reporter assay

A HR-based transient YFP reporter was constructed based on the pA7-YFP vector. The 1-510

bp and 229-720 bp coding sequences of YFP were cloned by PCR and fused together with an

18 bp linker (GGATCC ACTAGT GTCGAC), creating a split YFP with 282 bp overlapping.

The isolation and PEG transformation of Arabidopsis mesophyll protoplasts were as

described2. The transformed protoplasts were examined using a flow cytometer (BECKMAN

COULTER MoFloTM XDP, USA) after 16-24 hours of incubation in the dark according to the

manufacturer’s instructions.

Generation of Arabidopsis and rice stable transgenic plants

The pCambia1300 vectors containing the hSpCas9 expression cassette and the guide RNA

expression cassettes were transformed into Agrobacterium strain GV3101 and EHA105 by

the freeze-thaw method for transformation of Arabidopsis and rice, respectively. Healthy

Arabidopsis Col-0 wild type plants at the flowering stage were used for transformation by the

floral dipping method3. The collected seeds were screened on MS plates with 20 g/L

hygromycin. Agrobacterium-mediated transformation of the callus of rice cultivar Kasalath

was conducted as described4.

RFLP analysis of genome modification

Genomic DNA was extracted from stable transgenic plants from hygromycin selection and

wild type control plants. PCR was performed using specific primers for each target (Table S2).

After purification, about 400 nanograms of PCR product was digested overnight with the

corresponding restriction enzymes designed for each target site. Digested DNA was separated

on an ethidium bromide-stained agarose gel (1.5%). The digest-resistant bands were

recovered and cloned into the pZeroBack Blunt vector (Tiangen Biotech, China), and

mutations were identified by Sanger sequencing of individual clones.

SUPPLEMENTARY NOTE

Sequence of the sgRNA and Cas9 expression cassettes

>AtU6-26 sgRNA

GGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTAA

GCTTCGTTGAACAACGGAAACTCGACTTGCCTTCCGCACAATACATCATTTCTTCTT

AGCTTTTTTTCTTCTTCTTCGTTCATACAGTTTTTTTTTGTTTATCAGCTTACATTTTC

TTGAACCGTAGCTTTCGTTTTCTTCTTTTTAACTTTCCATTCGGAGTTTTTGTATCTT

GTTTCATAGTTTGTCCCAGGATTAGAATGATTAGGCATCGAACCTTCAAGAATTTGA

TTGAATAAAACATCTTCATTCTTAAGATATGAAGATAATCTTCAAAAGGCCCCTGGG

AATCTGAAAGAAGAGAAGCAGGCCCATTTATATGGGAAAGAACAATAGTATTTCTT

ATATAGGCCCATTTAAGTTGAAAACAATCTTCAAAAGTCCCACATCGCTTAGATAA

GAAAACGAAGCTGAGTTTATATACAGCTAGAGTCGAAGTAGTGATTGGGTCTTCGA

GAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA

ACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTCCCTTCGAAGGGCCTTTCT

CAGATATCCATCACACTGGCGGCCGCTCGAGGTCGACGGTATCGATAAGCTT

The AtU6-26 sequence and sgRNA are highlighted in magenta and yellow, respectively.

>OsU6-2 sgRNA

GGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTG

GATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGA

AGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGA

GGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGG

CGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTAT

AAACCCGCGCGCTGTCGCTTGTGTGGGTCTTCGAGAAGACCTGTTTTAGAGCTAG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA

GTCGGTGCTTTTTTTGTCCCTTCGAAGGGCAATTCTGCAGATATCCATCACACTGGC

GGCCGCTCGAGGTCGACGGTATCGATAAGCTT

The OsU6-2 sequence and sgRNA are highlighted in green and yellow, respectively.

>2×35S-Cas9-Nos

AAGCTTGCATGCCTGCAGGTCAACATGGTGGAGCACGACACACTTGTCTACTCCA

AAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACA

AAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTAT

TGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAA

GGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCC

CACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGC

AAGTGGATTGATGTGATAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAAT

ATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGG

TGATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGA

AGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAA

GGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCC

ACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTG

GATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCG

CAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACCTCGACCTC

AACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATT

GCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCA

TTTACGAACGATACTCGAGATGGACTATAAGGACCACGACGGAGACTACAAGGAT

CATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGA

AGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGA

CATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCC

AGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAAC

CTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTG

AAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTG

CAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGAC

TGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTT

CGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCAC

CTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATC

TGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCT

GAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC

AACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCC

ATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGC

TGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGG

CCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAG

CTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGC

GACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGC

TGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTC

TATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTC

GTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGA

ACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTT

CATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTG

AACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCC

CACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTT

ACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCAT

CCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACC

AGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAG

GGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGC

CCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTA

TAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTC

CTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGG

AAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTC

GACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT

ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAA

CGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAG

ATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGA

AGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGA

TCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTC

CGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACC

TTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACG

AGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGAC

AGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAA

CATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAA

CAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCA

GATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTA

CCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCA

ACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGAC

GACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGC

GACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAG

CTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCG

AGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGG

TGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAA

CACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTG

AAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCG

AGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAAC

CGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTAC

AAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAG

GCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGAT

TACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGA

AACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTG

CTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCT

TCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAA

GAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTAT

TCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGT

GTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATC

CCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCAT

CAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTG

GCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATG

TGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGA

TAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATC

ATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGG

ACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGC

CGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCA

AGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCT

GGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGAC

CTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAG

GCAAAAAAGAAAAAGTAAGGATCCTGATTGATCGATAGAGCTCGAATTTCCCCGAT

CGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCG

ATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATG

CATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAA

TACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGT

GTCATCTATGTTACTAGATCGGGAATTC

The 2x35S, 3xFLAG, NLS, hSpCas9 and Nos terminator sequences are highlighted,

respectively.

REFERENCES

1. Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science

339, 819-823 (2013).

2. Yoo, S.D., Cho, Y.H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell

system for transient gene expression analysis. Nature protocols 2, 1565-1572 (2007).

3. Clough, S.J. & Bent, A.F. Floral dip: a simplified method for

Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal

16, 735-743 (1998).

4. Hiei, Y., Ohta, S., Komari, T. & Kumashiro, T. Efficient transformation of rice (Oryza

sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the

T-DNA. The Plant Journal 6, 271-282 (1994).

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