Supplementary information, Figure S1

Transcription

1 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.

2 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.

3 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.

4 Supplementary information, Figure S4 Targeted indel mutations induced by engineered sgrna:cas9 at the BRI1 gene sgrna2 site in Arabidopsis. Alleles shown were amplified from genomic DNA isolated from 3 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). The number of clones representing each mutant allele is shown in brackets.

5 Supplementary information, Figure S5 Targeted indel mutations induced by engineered sgrna:cas9 at the BRI1 gene sgrna3 site in Arabidopsis. Alleles shown were amplified from genomic DNA isolated from 4 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). The number of clones representing each mutant allele is shown in brackets.

6 Supplementary information, Figure S6 Targeted indel mutations induced by engineered sgrna:cas9 at the GAI gene sgrna1 site in Arabidopsis. Alleles shown were amplified from genomic DNA isolated from 3 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). The number of clones representing each mutant allele is shown in brackets.

7 Supplementary information, Figure S7 Targeted indel mutations induced by engineered sgrna:cas9 at the JAZ1 gene sgrna1 site in Arabidopsis. Alleles shown were amplified from genomic DNA isolated from 5 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). The number of clones representing each mutant allele is shown in brackets.

8 Supplementary information, Figure S8 Targeted indel mutations induced by engineered sgrna:cas9 at the ROC5 gene sgrna1 site in rice. Alleles shown were amplified from genomic DNA isolated from 5 independent T0 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). The number of clones representing each mutant allele is shown in brackets.

9 Supplementary information, Figure S9 Targeted indel mutations induced by engineered sgrna:cas9 at the SPP gene sgrna1 site in rice. Alleles shown were amplified from genomic DNA isolated from 1 independent T0 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). The number of clones representing each mutant allele is shown in brackets.

10 Supplementary information, Figure S10 Targeted indel mutations induced by engineered sgrna:cas9 at the YSA gene sgrna1 site in rice. Alleles shown were amplified from genomic DNA isolated from 12 independent T0 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). The number of clones representing each mutant allele is shown in brackets. The plants #5 and #7 correspond to the two plants showing albino leaf phenotype.

11 Supplementary information, Figure S11 Targeted indel mutations induced by engineered sgrna:cas9 at the YSA gene sgrna2 site in rice. Alleles shown were amplified from genomic DNA isolated from 6 independent T0 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). The number of clones representing each mutant allele is shown in brackets. The plants #5 and #6 correspond to the two plants showing albino leaf phenotype.

12 Supplementary information, Table S1 Mutation detected in the T1 transgenic plants of Arabidopsis Plant ID No. of clones No. of clones with No. of different sequenced mutant alleles detected mutant alleles detected BRI1 sgrna Total BRI1 sgrna Total BRI1 sgrna Total GAI sgrna Total JAZ1 sgrna Total

13 Supplementary information, Table S2 Mutation detected in the T0 transgenic plants of rice Plant ID No. of clones No. of clones with No. of different sequenced mutant alleles detected mutant alleles detected ROC5 sgrna Total SPP sgrna Total YSA sgrna Total YSA sgrna Total

14 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 AtU F TTATTTTAACTTGCTATTTCTAGCTCTAAAACAGGTCTTCTC GAAGACCCAATCACTACTTCGACTCTAGCTGTA AtU R 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

15 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 px260 1 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 px330 1 by PCR using AtU F and AtU R (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 bp and bp coding sequences of YFP were cloned by PCR and fused together with an

16 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 described 2. The transformed protoplasts were examined using a flow cytometer (BECKMAN COULTER MoFlo TM XDP, USA) after 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 method 3. 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 described 4. 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.

17 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

18 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

19 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

20 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, (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, (2007). 3. Clough, S.J. & Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, (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, (1994).