A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex targeting in plants · 2015-08-01 · 1 Supplementary Information A robust CRISPR/Cas9 system for convenient,

1

Supplementary Information

A robust CRISPR/Cas9 system for convenient, high-efficiency

multiplex targeting in plants

Xingliang Ma1,4, Qunyu Zhang1,2,4, Qinlong Zhu2,4,Wei Liu1,2,4, Yan Chen5, Rong

Qiu1,2,4, Bin Wang1,2,4, Zhongfang Yang1,2,4, Heying Li1,2,4, Yuru Lin1,2,4, Yongyao

Xie1,2,4, Rongxin Shen1,2,4, Shuifu Chen1,2,4, Zhi Wang4, Yuanling Chen1,2,4, Jingxing

Guo1,2,4, Letian Chen1,2,3,4,Xiucai Zhao1,2,4 Zhicheng Dong5 & Yao-Guang Liu1,2,4*

1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources

2 Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial

Higher Education Institutions 3 Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural

Organisms. 4 College of Life Sciences, South China Agricultural University, Guangzhou 510642, China

5 Key Laboratory of South China Agriculture Plant Molecular Analysis and Genetic

Improvement, South China Botanical Garden, Chinese Academy of Sciences

*Correspondence: Yao-Guang Liu ([email protected])

Contents Page

Supplementary Figure 1 Sequence of Cas9p 2-3

Supplementary Figure 2 Distributions of GC contents in six Cas9 genes 3

Supplementary Figure 3 Sequences of the sgRNA expression cassettes 4-9

Supplementary Figure 4 Procedures for generation of a sgRNA expression cassette containing

a target sequence by overlapping PCR.

9

Supplementary Figure 5 Examples of direct sequencing of PCR products containing targeted

sites in rice T1 plants.

Supplementary Figure 6 Examples of direct sequencing of PCR products containing targeted

sites in Arabidopsis T1 plants.

10

11

Supplementary Figure 7 Secondary structures of target-sgRNAs

Supplementary Table 1. Primers used for Golden Gate cloning of sgRNA expression cassettes.

12

13

Supplementary Table 2. Primers used for Gibson Assembly of sgRNA expression cassettes. 13-14

Supplementary Table 3. Primers used for introduction of target sequences into sgRNA

expression cassettes by overlapping PCR.

14

Supplementary Table 4. Summary of the binary constructs , target sequences, and editing rates. 15-16

Supplementary Table 5. Targeted genomic mutations in T0 plants of rice and T1 plants of

Arabidopsis.

17-31

Supplementary Table 6. Inheritance and genetic segregation of the edited sites in rice T1 lines.

Supplementary Table 7. Inheritance of the edited sites in Arabidopsis T2 lines.

Supplementary Table 8. Primers used for expression analysis .

31

32

32-33

2

Supplementary Figures

1 ATGGCTCCTA AGAAGAAGCG GAAGGTTGGT ATTCACGGGG TGCCTGCGGC TGACAAGAAG

61 TACTCCATCG GCCTCGACAT CGGCACCAAC AGCGTCGGCT GGGCGGTGAT CACCGACGAG

121 TACAAGGTCC CGTCCAAGAA GTTCAAGGTC CTGGGCAACA CCGACCGCCA CTCCATCAAG

181 AAGAACCTCA TCGGCGCCCT CCTCTTCGAC TCCGGCGAGA CGGCGGAGGC GACCCGCCTC

241 AAGCGCACCG CCCGCCGCCG CTACACCCGC CGCAAGAACC GCATCTGCTA CCTCCAGGAG

301 ATCTTCTCCA ACGAGATGGC GAAGGTCGAC GACTCCTTCT TCCACCGCCT CGAGGAGTCC

361 TTCCTCGTGG AGGAGGACAA GAAGCACGAG CGCCACCCCA TCTTCGGCAA CATCGTCGAC

421 GAGGTCGCCT ACCACGAGAA GTACCCCACT ATCTACCACC TTCGTAAGAA GCTTGTTGAC

481 TCTACTGATA AGGCTGATCT TCGTCTCATC TACCTTGCTC TCGCTCACAT GATCAAGTTC

541 CGTGGTCACT TCCTTATCGA GGGTGACCTT AACCCTGATA ACTCCGACGT GGACAAGCTC

601 TTCATCCAGC TCGTCCAGAC CTACAACCAG CTCTTCGAGG AGAACCCTAT CAACGCTTCC

661 GGTGTCGACG CTAAGGCGAT CCTTTCCGCT AGGCTCTCCA AGTCCAGGCG TCTCGAGAAC

721 CTCATCGCCC AGCTCCCTGG TGAGAAGAAG AACGGTCTTT TCGGTAACCT CATCGCTCTC

781 TCCCTCGGTC TGACCCCTAA CTTCAAGTCC AACTTCGACC TCGCTGAGGA CGCTAAGCTT

841 CAGCTCTCCA AGGATACCTA CGACGATGAT CTCGACAACC TCCTCGCTCA GATTGGAGAT

901 CAGTACGCTG ATCTCTTCCT TGCTGCTAAG AACCTCTCCG ATGCTATCCT CCTTTCGGAT

961 ATCCTTAGGG TTAACACTGA GATCACTAAG GCTCCTCTTT CTGCTTCCAT GATCAAGCGC

1021 TACGACGAGC ACCACCAGGA CCTCACCCTC CTCAAGGCTC TTGTTCGTCA GCAGCTCCCC

1081 GAGAAGTACA AGGAGATCTT CTTCGACCAG TCCAAGAACG GCTACGCCGG TTACATTGAC

1141 GGTGGAGCTA GCCAGGAGGA GTTCTACAAG TTCATCAAGC CAATCCTTGA GAAGATGGAT

1201 GGTACTGAGG AGCTTCTCGT TAAGCTTAAC CGTGAGGACC TCCTTAGGAA GCAGAGGACT

1261 TTCGATAACG GCTCTATCCC TCACCAGATC CACCTTGGTG AGCTTCACGC CATCCTTCGT

1321 AGGCAGGAGG ACTTCTACCC TTTCCTCAAG GACAACCGTG AGAAGATCGA GAAGATCCTT

1381 ACTTTCCGTA TTCCTTACTA CGTTGGTCCT CTTGCTCGTG GTAACTCCCG TTTCGCTTGG

1441 ATGACTAGGA AGTCCGAGGA GACTATCACC CCTTGGAACT TCGAGGAGGT TGTTGACAAG

1501 GGTGCTTCCG CCCAGTCCTT CATCGAGCGC ATGACCAACT TCGACAAGAA CCTCCCCAAC

1561 GAGAAGGTCC TCCCCAAGCA CTCCCTCCTC TACGAGTACT TCACGGTCTA CAACGAGCTC

1621 ACCAAGGTCA AGTACGTCAC CGAGGGTATG CGCAAGCCTG CCTTCCTCTC CGGCGAGCAG

1681 AAGAAGGCTA TCGTTGACCT CCTCTTCAAG ACCAACCGCA AGGTCACCGT CAAGCAGCTC

1741 AAGGAGGACT ACTTCAAGAA GATCGAGTGC TTCGACTCCG TCGAGATCAG CGGCGTTGAG

1801 GACCGTTTCA ACGCTTCTCT CGGTACCTAC CACGATCTCC TCAAGATCAT CAAGGACAAG

1861 GACTTCCTCG ACAACGAGGA GAACGAGGAC ATCCTCGAGG ACATCGTCCT CACTCTTACT

1921 CTCTTCGAGG ATAGGGAGAT GATCGAGGAG AGGCTCAAGA CTTACGCTCA TCTCTTCGAT

1981 GACAAGGTTA TGAAGCAGCT CAAGCGTCGC CGTTACACCG GTTGGGGTAG GCTCTCCCGC

2041 AAGCTCATCA ACGGTATCAG GGATAAGCAG AGCGGCAAGA CTATCCTCGA CTTCCTCAAG

2101 TCTGATGGTT TCGCTAACAG GAACTTCATG CAGCTCATCC ACGATGACTC TCTTACCTTC

2161 AAGGAGGATA TTCAGAAGGC TCAGGTGTCC GGTCAGGGCG ACTCTCTCCA CGAGCACATT

2221 GCTAACCTTG CTGGTTCCCC TGCTATCAAG AAGGGCATCC TTCAGACTGT TAAGGTTGTC

2281 GATGAGCTTG TCAAGGTTAT GGGTCGTCAC AAGCCTGAGA ACATCGTCAT CGAGATGGCT

2341 CGTGAGAACC AGACTACCCA GAAGGGTCAG AAGAACTCGA GGGAGCGCAT GAAGAGGATT

2401 GAGGAGGGTA TCAAGGAGCT TGGTTCTCAG ATCCTTAAGG AGCACCCTGT CGAGAACACC

2461 CAGCTCCAGA ACGAGAAGCT CTACCTCTAC TACCTCCAGA ACGGTAGGGA TATGTACGTT

2521 GACCAGGAGC TCGACATCAA CAGGCTTTCT GACTACGACG TCGACCACAT TGTTCCTCAG

2581 TCTTTCCTTA AGGATGACTC CATCGACAAC AAGGTCCTCA CGAGGTCCGA CAAGAACAGG

2641 GGTAAGTCGG ACAACGTCCC TTCCGAGGAG GTTGTCAAGA AGATGAAGAA CTACTGGAGG

2701 CAGCTTCTCA ACGCTAAGCT CATTACCCAG AGGAAGTTCG ACAACCTCAC GAAGGCTGAG

2761 AGGGGTGGCC TTTCCGAGCT TGACAAGGCT GGTTTCATCA AGAGGCAGCT TGTTGAGACG

2821 AGGCAGATTA CCAAGCACGT TGCTCAGATC CTCGATTCTA GGATGAACAC CAAGTACGAC

2881 GAGAACGACA AGCTCATCCG CGAGGTCAAG GTGATCACCC TCAAGTCCAA GCTCGTCTCC

2941 GACTTCCGCA AGGACTTCCA GTTCTACAAG GTCCGCGAGA TCAACAACTA CCACCACGCT

3001 CACGATGCTT ACCTTAACGC TGTCGTTGGT ACCGCTCTTA TCAAGAAGTA CCCTAAGCTT

3061 GAGTCCGAGT TCGTCTACGG TGACTACAAG GTCTACGACG TTCGTAAGAT GATCGCCAAG

3

3121 TCCGAGCAGG AGATCGGCAA GGCCACCGCC AAGTACTTCT TCTACTCCAA CATCATGAAC

3181 TTCTTCAAGA CCGAGATCAC CCTCGCCAAC GGCGAGATCC GCAAGCGCCC TCTTATCGAG

3241 ACGAACGGTG AGACTGGTGA GATCGTTTGG GACAAGGGTC GCGACTTCGC TACTGTTCGC

3301 AAGGTCCTTT CTATGCCTCA GGTTAACATC GTCAAGAAGA CCGAGGTCCA GACCGGTGGC

3361 TTCTCCAAGG AGTCTATCCT TCCAAAGAGA AACTCGGACA AGCTCATCGC TAGGAAGAAG

3421 GATTGGGACC CTAAGAAGTA CGGTGGTTTC GACTCCCCTA CTGTCGCCTA CTCCGTCCTC

3481 GTGGTCGCCA AGGTGGAGAA GGGTAAGTCG AAGAAGCTCA AGTCCGTCAA GGAGCTCCTC

3541 GGCATCACCA TCATGGAGCG CTCCTCCTTC GAGAAGAACC CGATCGACTT CCTCGAGGCC

3601 AAGGGCTACA AGGAGGTCAA GAAGGACCTC ATCATCAAGC TCCCCAAGTA CTCTCTTTTC

3661 GAGCTCGAGA ACGGTCGTAA GAGGATGCTG GCTTCCGCTG GTGAGCTCCA GAAGGGTAAC

3721 GAGCTTGCTC TTCCTTCCAA GTACGTGAAC TTCCTCTACC TCGCCTCCCA CTACGAGAAG

3781 CTCAAGGGTT CCCCTGAGGA TAACGAGCAG AAGCAGCTCT TCGTGGAGCA GCACAAGCAC

3841 TACCTCGACG AGATCATCGA GCAGATCTCC GAGTTCTCCA AGCGCGTCAT CCTCGCTGAC

3901 GCTAACCTCG ACAAGGTCCT CTCCGCCTAC AACAAGCACC GCGACAAGCC CATCCGCGAG

3961 CAGGCCGAGA ACATCATCCA CCTCTTCACG CTCACGAACC TCGGCGCCCC TGCTGCTTTC

4021 AAGTACTTCG ACACCACCAT CGACAGGAAG CGTTACACGT CCACCAAGGA GGTTCTCGAC

4081 GCTACTCTCA TCCACCAGTC CATCACCGGT CTTTACGAGA CTCGTATCGA CCTTTCCCAG

4141 CTTGGTGGTG ATAAGCGTCC TGCTGCCACC AAAAAGGCCG GACAGGCTAA GAAAAAGAAG

4201 TAG

Supplementary Figure 1. Sequence of Cas9p. The nucleotides encoding the nuclear

localization signals are shown in red.

Supplementary Figure 2. Distributions of GC contents in six Cas9 genes, Cas9p (this study),

Cas9-CG (Shan et al., 2013), Cas9-JS (Li et al., 2013), Cas9-YY (Xie and Yang 2013), Cas9-JZ

(Mao et al., 2013), and Cas9-YB (Zhou et al., 2014), used for genome targeting in plants. The

GC content distributions were scanned in 100-nt sliding windows with a 10-nt overlap between

windows.

Cas9p

Cas9-YYCas9-JZ

Cas9-BYCas9-CG

Cas9-JS

Gene coordinate (nt, 5’—3’)

4

Supplementary Figure 3. Sequences of the sgRNA vectors and those of the expression cassettes

PCR product of LacZ-U6a-sgRNA (839 bp, 809 bp after Bsa I digestion)

TTCAGAGGTCTCTctcgACTAGTATGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAA

GGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgca

gcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctga

atggctaaTTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGA

ACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTAC

AGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTC

CTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGT

GCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCA

CCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCG(19-20 bp

target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT

GGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

PCR product of OsU6a-sgRNA (629 bp, 599 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCC

GAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGC

CCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGAC

GATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCC

GGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGT

AGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTG

GCTTGGCTGCCG(19-20 bp target)GTTTTAG

AGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAA

GAGCTT GGAGTGGATGGNNNNNNNCGAGACCCACGCT

acccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC

ACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGACGCGTTGA

Mlu I

E.coli Promoter LacZ (alpha)

CATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttac

ccaacttaa tcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagtt

gcgcagcctgaatggctaaTTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGT

GAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTAT

CGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTAT

GGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCA

CTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTAT

ATGCGCGGGTGCTGGCTTGGCTGCCGAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone）

LacZ-OsU6a-sgRNA structure in the plasmid

BamH I Bsa I

Hind IIIBsa I


ACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGATTTTTTCC

TGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTA

AAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTA

GGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGT

TGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGA

TGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCGA

GAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone）Hind IIIBsa I

BamH I Bsa I

OsU6a-sgRNA structure in the plasmid

5

PCR product of OsU6b-sgRNA (515 bp, 485 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGATGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCAC

ATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGA

GAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGCCGACGAGCGTGTACTACCGCGCGGGATGCC

GCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGGAGACAGGTTTAGTACCACCTC

GCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTG(19-20 bp

target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT

AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGA

CCCACG CT

PCR product of OsU6c-gRNA (924 bp, 894 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGActcattagcggtatgcatgttggtagaagtcggagatgta

aataattttcattatataaaaaaggtacttcgagaaaaataaatgcatacgaattaattctttttatgttttttaaacc

aagtatatagaatttattgatggttaaaatttcaaaaatatgacgagagaaaggttaaacgtacggcatatacttctga

acagagagggaatatggggtttttgttgctcccaacaattcttaagcacgtaaaggaaaaaagcacattatccacattg

tacttccagagatatgtacagcattacgtaggtacgttttctttttcttcccggagagatgatacaataatcatgtaaa

cccagaatttaaaaaatattctttactataaaaattttaattagggaacgtattattttttacatgacaccttttgaga

aagagggacttgtaatatgggacaaatgaacaatttctaagaaatgggcatatgactctcagtacaatggaccaaattc

cctccagtcggcccagcaatacaaagggaaagaaatgagggggcccacaggccacggcccacttttctccgtggtgggg

agatccagctagaggtccggcccacaagtggcccttgccccgtgggacggtgggattgcagagcgcgtgggcggaaaca

acagtttagtaccacctcgctcacgcaacgacgcgaccacttgcttataagctgctgcgctgaggctcaG(19-20 bp

target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

TGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

acccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGA

TGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATG

GGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGAGAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGC

CCACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGA

GGGGAGACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTGAGA

GACCTCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone)

OsU6b-sgRNA structure in the plasmid



ctcattagcggtatgcatgttggtagaagtcggagatgtaaataattttcattatataaaaaaggtacttcgagaaaaataaat

gcatacgaattaattctttttatgttttttaaaccaagtatatagaatttattgatggttaaaatttcaaaaatatgacgagag

aaaggttaaacgtacggcatatacttctgaacagagagggaatatggggtttttgttgctcccaacaattcttaagcacgtaaa

ggaaaaaagcacattatccacattgtacttccagagatatgtacagcattacgtaggtacgttttctttttcttcccggagaga

tgatacaataatcatgtaaacccagaatttaaaaaatattctttactataaaaattttaattagggaacgtattattttttaca

tgacaccttttgagaaagagggacttgtaatatgggacaaatgaacaatttctaagaaatgggcatatgactctcagtacaatg

gaccaaattccctccagtcggcccagcaatacaaagggaaagaaatgagggggcccacaggccacggcccacttttctccgtgg

tggggagatccagctagaggtccggcccacaagtggcccttgccccgtgggacggtgggattgcagagcgcgtgggcggaaaca

acagtttagtaccacctcgctcacgcaacgacgcgaccacttgcttataagctgctgcgctgaggctcagAGAGACCTCTGAAG

ATAACATACTAAGCTTggcact(pUC18 backbone)

OsU6c-sgRNA structure in the plasmid

6

LacZ-OsU3-sgRNA structure in the plasmid

PCR product of LacZ-OsU3-sgRNA (774 bp, 744 bp after Bsa I digestion)


GGAGGCAGCTATGctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgca


atggcTAAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCAT

GGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGC

TGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTT

CGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGAC

TAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA (19-

20 target）GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGC

T

PCR product of OsU3-sgRNA (603 bp, 573 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAA

GGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGG

AGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCG

GGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGG

CCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAG

TACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA(19-20 bp

target) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG

GCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCG

AGACCC ACGCT



AGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGA

ATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTG

GGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTC

AGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCAC

ATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCAAGAGACCTCTGAAGATAACATACTAAGCTTggcact(p

UC18 backbone)

OsU3-sgRNA structure in the plasmid

7

PCR product of LacZ-AtU3b-sgRNA (709 bp, 679 bp after Bsa I digestion)


GGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgca


atggctaatttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaag

aagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcga

gcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtaccca

tgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactga

gggagcaacattggtcA(19-20 bp target)GTTTTAGAGCTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT

TGGAGT GGATGGNNNNNNNCGAGACCCACGCT

PCR product of LacZ-AtU3b-sgRNA (507 bp, 477 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAtttactttaaattttttcttatgcagcctgtgatggataact

gaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaact

tcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagatt

cactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaa

tcttgttgtatatataacactgagggagcaacattggtcA(19-20 bp

target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC

TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

acccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATC

GGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttaca

acgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaa

tagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaatttactttaaattttttctt

atgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacga

aacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatg

taatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctccta

atttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcaAGAGACC

TCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone)

LacZ-AtU3b-sgRNA structure in the plasmid



GGCAGCAAAGGAtttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggt

ttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagctta

tatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgccca

gtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatat

aacactgagggagcaacattggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone)

AtU3b-sgRNA structure in the plasmid

8

PCR product of LacZ-AtU3d-sgRNA (494 bp, 464 bp after Bsa I digestion)

TTCAGAGGTCTCTctcgACTAGTATGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAG

GAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagca

catccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggc

taaataagcttatgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatat

atggctacaaagcaccattggtca (19-20 bp target)

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

PCR product of AtU3d-sgRNA (284 bp, 254 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAataagcttatgatttcttttttcttacgaattttgcgtcc

cacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtcA(19-20 bp

target)GTTTTAGAGCTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT

TGGAGT GGATGGNNNNNNNCGAGACCCACGCT

PCR product of AtU6-1-sgRNA (487 bp, 457 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAAGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGA

TCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGC

ATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTT

TTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTA

TATATGGTTAGAGACGAAGTAGTGATTG(19-20 bp

target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA

CCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT


ACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAataagctt

atgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccat

tggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone）

AtU3d-sgRNA structure in the plasmid

9

PCR product of AtU6-29-sgRNA (502 bp, 472 bp after Bsa I digestion) TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAaaatatcagagatctcttacagttagtttcgttcttaatc

caaactactgcagcctgacagacaaatgaggatgcaaacaattttaaagtttatctaacgctagctgttttgtttcttc

tctctggtgcaccaacgacggcgttttctcaatcataaagaggcttgttttacttaaggccaataatgttgatggatcg

aaagaagagggcttttaataaacgagcccgtttaagctgtaaacgatgtcaaaaacatcccacatcgttcagttgaaaa

tagTagctctgtttatatattggtagagtcgactaagagattG(19-20 bp

target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA

CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

Supplementary Figure 4. Procedures for generation of a sgRNA expression cassette

containing a target sequence by overlapping PCR. The chimeric primers with target sequence

strands are given in Supplementary Table 1. The first PCR is carried out in two separated

reactions with U-F/U#T#- and gRT#+/gR-R primer pair, respectively, or in one reaction with

the all the 4 primers. U# indicates a given promoter, and T#+ and T#- indicate forward and

reverse strands of a target sequence, respectively.



GGCAGCAAAGGAaaatatcagagatctcttacagttagtttcgttcttaatccaaactactgcagcctgacagacaaa

tgaggatgcaaacaattttaaagtttatctaacgctagctgttttgtttcttctctctggtgcaccaacgacggcgtt

ttctcaatcataaagaggcttgttttacttaaggccaataatgttgatggatcgaaagaagagggcttttaataaacg

agcccgtttaagctgtaaacgatgtcaaaaacatcccacatcgttcagttgaaaatagTagctctgtttatatattgg

tagagtcgactaagagattgAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone)

AtU6-29-sgRNA structure in the plasmid

Extension

2nd PCR (overlapping PCR)Annealing

PpsBsa I

Bsa I

Bsa I Bsa I

PCR

U-F

gR-R

Vector backboneU3/U6 promoter

sgRNA region

Target sequence (-)

Target sequence (+)

1st PCR

Pgs

1st PCR

U#T#-

gRT#+

10

Supplementary Figure 5. Examples of direct sequencing of PCR products containing targeted

sites in rice T1 plants. The sequencing chromatograms with overlapping traces were decoded using

the DSD method (Ma et al., 2015). The plant numbers (Os #) are the same as in Supplementary

Table 4. Arrows indicate the start positions at where or from where the mutations occurred.

11

Supplementary Figure 6. The secondary structures of target-sgRNAs for Os02g0700600 that did

not have successful editing (0/5), OsFTL7 and OsFTL8 with high editing rates (22/26, 22/26). The

secondary structures were analyzed using the program RNA Folding Form

(http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3).

Target-sgRNA:

(20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA

CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

Target sequence

Os02g0700600 OsFTL8OsFTL7

Target sequence

Target sequence

12

Supplementary Figure 7. Examples of direct sequencing of PCR products containing targeted

sites in Arabidopsis T1 plants. The sequencing chromatograms with overlapping trances were

decoded using the DSD method (Ma et al., 2015). The plant numbers (At #) are the same as in

Supplementary Table 4. Arrows indicate the positions at where or from where the mutations

occurred. Two 7-bp direct repeats (in blue) that flank the target site as indicated in At 4-2 might

mediate the identical 30-bp deletion in At 4-2, At 4-17 and At 4-44 (see Supplementary Table 4).

13

Supplementary Table 1. Primers used for introduction of target sequences into sgRNA

expression cassettes by overlapping PCR.

Primer Sequence

gRT#+ (N19 or N20)gttttagagctagaaat-3’

OsU6aT#- (N19 or N20)Cggcagccaagccagca-3’

OsU6bT#- (N19 or N20)Caacacaagcggcagc-3’

OsU6cT#-

OsU3T#-

(N19 or N20)Ctgagcctcagcgcag-3’

(N19 or N20)Tgccacggatcatctgc-3

AtU3bT#- (N19 or N20)Tgaccaatgttgctcc-3’

AtU3dT#- (N19 or N20)Tgaccaatggtgctttg-3’

AtU6-1T#- (N19 or N20)Caatcactacttcgtct-3

AtU6-29T#- (N19 or N20)Caatctcttagtcgact-3’

Note: For regular targets, 19 nucleotides (N19) are used as the target sequences that do not

include the first base G or A; for irregular targets, 20 nucleotides (N20) are used as the target

sequences.

Supplementary Table 2. Primers used for Golden Gate cloning of sgRNA expression

cassettes.

Position Primer Sequence (5’--3’)

1st

PCR U-F CTCCGTTTTACCTGTGGAATCG

gR-R CGGAGGAAAATTCCATCCAC

2nd

PCR

Site B-L

Site 2

Pps-GGL

TTCAGAggtctcTctcgACTAGTATGGAATCGGCAGCAAAGG

Pgs-GG2 AGCGTGggtctcGtcagggTCCATCCACTCCAAGCTC

Site 2

Site 3

Pps-GG2 TTCAGAggtctcTctgacacTGGAATCGGCAGCAAAGG

Pgs-GG3 AGCGTGggtctcGtcttcacTCCATCCACTCCAAGCTC

Site 3

Site 4

Pps-GG3 TTCAGAggtctcTaagacttTGGAATCGGCAGCAAAGG

Pgs-GG4 AGCGTGggtctcGagtccttTCCATCCACTCCAAGCTC

Site 4

Site 5

Pps-GG4 TTCAGAggtctcTgactacaTGGAATCGGCAGCAAAGG

Pgs-GG5 AGCGTGggtctcGgtccacaTCCATCCACTCCAAGCTC

Site 5

Site 6

Pps-GG5 TTCAGAggtctcTggacttgTGGAATCGGCAGCAAAGG

Pgs-GG6 AGCGTGggtctcGcagatagTCCATCCACTCCAAGCTC

Site 6

Site 7

Pps-GG6 TTCAGAggtctcTtctgcaaTGGAATCGGCAGCAAAGG

Pgs-GG7 AGCGTGggtctcGacctcaaTCCATCCACTCCAAGCTC

Site 7

Site 8

Pps-GG7 TTCAGAggtctcTaggtttcTGGAATCGGCAGCAAAGG

Pgs-GG8 AGCGTGggtctcGagcgttcTCCATCCACTCCAAGCTC

Site 8

Site B-R

Pps-GG8 TTCAGAggtctcTcgctgatTGGAATCGGCAGCAAAGG

Pgs-GGR AGCGTGggtctcGaccgACGCGTATCCATCCACTCCAAGCTC

Flanking

Primers

PB-L

PB-R

GCGCGCgGTctcGCTCGACTAGTATGG

GCGCGCggtctcTACCGACGCGTATCC

Note:

14

(1) One or multiple sgRNA expression cassettes are amplified with the primer pairs in this way:

1 cassette: Pps-GGL/Pgs-GGR;

2 cassettes: Pps-GGL/Pgs-GG2, Pps-GG2/Pgs-GGR;

3 cassettes: Pps-GGL/Pgs-GG2, Pps-GG2/Pgs-GG3, Pps-GG3/Pgs-GGR;

4 cassettes: Pps-GGL/Pgs-GG2, Pps-GG2/Pgs-GG3, Pps-GG3/Pgs-GG4, Pps-GG4/Pgs-GGR;

and so on.

(2) The Bsa I-cutting non-palindromic ends with the same color are compatible for ligation.

(3) ACTAGT and ACGCGT are Spe I and Mlu I sites, respectively.

(4) If more sgRNA expression cassettes are needed, new primer sets can be designed with distinct

Bsa I-cleaving sites.

(5) The flanking primers can be used to amplify, if necessary, the ligated sgRNA expression

cassettes. Lowercase letters are bases that mismatch with the linked binary vector flanking

sequences, see Figure 1B.

(6) The Spe I site can be used for cloning of a second group of sgRNA expression cassettes by

Gibson Assembly if the OsU3 promoter (with a Spe I site) is not used in the first group of

sgRNA expression cassettes. Otherwise, modified Pgs-GGR (AGCGTGggtctcGaccg

AtGCGTATCCATCCACTCCAAGCTC-3), and PB-R (GCGCGCggtctcTACCGAtGCGTATCC) if

necessary，is used for cloning of the first group sgRNA expression cassettes. Then the unique Mlu

I site at the upstream of LacZ is used for cloning (by Gibson Assembly) of the second group of

sgRNA expression cassettes (see below).

Supplementary Table 3. Primers used for Gibson Assembly of sgRNA expression cassettes.

Position Primer Sequence (5’--3’)

1st

PCR U-F CTCCGTTTTACCTGTGGAATCG

gR-R CGGAGGAAAATTCCATCCAC

2nd

PCR

Site B-L

Site 2

U-GAL

ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGG

Pgs-GA2 CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG

Site 2

Site 3

U-GA2 GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAGG

Pgs-GA3 CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTCTTG

Site 3

Site 4

U-GA3 CGCTATAGTGTCACCTAAATCGTATGCGTGGTGGAATCGGCAGCAAAGG

Pgs-GA4 GTCGCTAGTTATTGCTCAGCGGCCAAGCTCATCCACTCCAAGCTCTTG

Site 4

Site 5

U-GA4 GAGCTTGGCCGCTGAGCAATAACTAGCGACTGGAATCGGCAGCAAAGG

Pgs-GA5 CATCGTCGCCGTCCAGCTCGACCATTGAACATCCACTCCAAGCTCTTG

Site 5

Site 6

U-GA5 GTTCAATGGTCGAGCTGGACGGCGACGATGTGGAATCGGCAGCAAAGG

Pgs-GA6 GCTCCGAATACGACTCACTATAGGGTGACCATCCACTCCAAGCTCTTG

Site 6 Site

7

U-GA6 GGTCACCCTATAGTGAGTCGTATTCGGAGCTGGAATCGGCAGCAAAGG

Pgs-GA7 CTGAGGTTAACCCTCACTAAAGGGAAGCTCCATCCACTCCAAGCTCTTG

Site 7

Site 8

U-GA7 GGAGCTTCCCTTTAGTGAGGGTTAACCTCAGTGGAATCGGCAGCAAAGG

Pgs-GA8 CGTGGTATGCTAGTTATTGCTCAGCCTCGACATCCACTCCAAGCTCTTG

Site 8

Site B-R

U-GA8 GTCGAGGCTGAGCAATAACTAGCATACCACGTGGAATCGGCAGCAAAGG

Pgs-GAR TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCCAAGCTCTTG

Note:

15

(1) One or multiple sgRNA expression cassettes are amplified with the primer pairs in this way:

1 cassette: Pps-GAL/Pgs-GAR;

2 cassettes: Pps -GAL/Pgs-GA2, Pps-GA2/Pgs-GAR;

3 cassettes: Pps-GAL/Pgs-GA2, Pps-GA2/Pgs-GA3, Pps-GA3/Pgs-GAR;

4 cassettes: Pps-GAL/Pgs-GA2, Pps-GA2/Pgs-GA3, Pps-GA3/Pgs-GA4, Pps-GG4/Pgs-GAR;

and so on.

(2) The same flanking primers PB-L and PB-R shown in Supplementary Table 1 can be used to

amplify, if necessary, the ligated sgRNA expression cassettes.

(3) If a second group of sgRNA expression cassettes are added into the Spe I site (when OsU3 is not

used in the first group), U-GAL/Pgs-GAR needed to modified according to the flanking sequences

of the Spe I site upstream of LacZ of the used U3/U6 promoters.

(4) If a second group of sgRNA expression cassettes are added into the Mlu I site (see above),

U-GAL/ Pgs-GAR are changed to：

U-GAL2：ctcgACTAGTATGGAATCGGCAGCAAAGGACGCGTATGGAATCGGCAGCAAAGG-3

Pgs-GAR2: GAGCAATATAGTCCTACAATGTCAACGCGTATCCATCCACTCCAAGCTCTTG-3

Supplementary Table 4. Summary of the binary constructs, target sequences of the genes, and

mutation rates of the analyzed sites. The starting base G for the U6 promoters and A for the U3

promoters of the regular targets are shown in blue. The nucleotides within rectangles are those that

may pair with the sgRNA sequence to form stem-loop structures.

Constr

uct

Gene Target sequence (5’---3’) Promoter No. mutated sites

/No. sequenced sites

Os 1 OsFTL1 GTCGGCCGGCAGCCGGATGACGG OsU6a 3/3

OsFTL4 GTCACGAGGTAG-GGATCCTTTGG OsU6b 3/3

OsFTL5 GACGCGCGGCTTGCCGGCGACGG OsU6c 3/3

OsFTL6 ATCTTCACTAGCCATGTCAAGGG OsU3 3/3

OsFTL9 TATTGGTGGCACCGACCTGAGGG OsU6c 3/3

OsFTL10 GGCTTGCTTACAACTGCAGATGG OsU6a 3/3

OsFTL11 TCATGTCGTCGGCGATATCGTGG OsU6b 0/3

OsFTL13 CGGTGCTGATGAAGGGATCCAGG OsU3 3/3

Os 2 OsFTL7 TCGGCGCGTCGGCGCTGCTGAGG OsU6a 22/26

OsFTL8 CGGCGAACAGCCTGGTGCTGGGG OsU6b 22/24

OsFTL12 TGGCAAGGAGTTCCGTTCCTCGG OsU6c 22/24

Os 3 OsGSTU GGCGGCCGCTCTTCCGCGGGTGG OsU6a 5/5

OsMRP15 TGCTAAAACTGAAACTAGTAAGG OsU3 3/4

OsAnP GGAGGAGGACGCGGCCGCCGTGG OsU6c 4/4

Os 4 Waxy TGTGTGCTTACAGCCATGGCAGG OsU6a 2/3

Waxy GAGCCTCGAGTGCTGCCTGCAGG OsU3 2/2

Waxy GTCTGAATCTTTTTCACTGCAGG OsU6b 1/3

Os 5 Os05g0591600 AGAGGATCGGCTATAATACCTGG OsU3 4/4

Os 6 Os03g0126800 TGCACCTTGTATTTGCAGTTGGG OsU6a 13/13

16

Os03g0126800 AAATCCCAACTGCAAATACAAGG OsU3 13/13

Os 7 Os07g0409500 GCGAGCGAGAGTCATGGAGGCGG OsU6a 5/6

Os07g0409500 GCACGCCAAGGTCGACGACAAGG OsU6a 4/5

Os 8 Os07g0625500 GCGAGATCGAGCGATGGCGACGG OsU6a 3/4

Os07g0625500 GGCGCTCGCCAAGTTCGACAGGG OsU6a 3/4

Os 9 Os07g0261200 ATTTATCCGTTCATGTCGATGGG OsU3 2/14

Os07g0261200 GATACTCACGATGG-TGGCGCTGG OsU6a 18/19

Os 10 Os05g0543000 AACCTGATCACCAACCCACTCGG OsU3 1/1

Os11g0549665 ATCTGGTGGGGCTGATCGGGGGG OsU3 1/1

Os10g0548600 GTGGTGGCAGCGCTGGTCGGCGG OsU6a 1/1

Os 11 Os05g0312500 AAGATCCATGAGTTCAAGAAGGG OsU3 5/5

Os04g0668400 GCGGA-AGCCTCGACAGTCACCGG OsU6a 5/5

Os02g0700600 GAAGTTGGTGACGGGACTTTCGG OsU6c 0/5

Os 12 Os07g0411300 GTCGTCCATGGCCCCCAACCCGG OsU6a 10/10

Os10g0484800 ACCTTGGATTCGGCGATGCCAGG OsU6a 8/8

Os 13 Os04g0595000 CCTCGATCCTCCTCTGCAGAGGG OsU6b 1/1

Os 14 Os12g0242700 ATTGTGAACTATGCCAAGTCAGG OsU3 10/10

Os 15 Os03g0216800 GGA-GGCTCCATCGAATCGATCGG OsU6b 7/8

Os 16 Os02g0459600 GGCTATTGGATCGTGCACCAAGG OsU6b 2/2

Os01g0891000 TCACCTTGCAAACATGGCGCAGG OsU6a 2/2

Os 17 Os10g0413900 CCTCGCCGCCGTACGTGTAGCGG OsU6a 8/10

Os10g0413900 GGGCGAGGGGATGGCGGCACCGG OsU6b 9/10

Os 18 Os06g0142000 TTCATGCTCAGGCATCGCTATGG OsU3 3/4

Os06g0142100 CATGGGGAACTTGGACTTGGCGG OsU6a 2/2

Os 19 Os03g0247300 CGCAAGACAGGGCACACCGGAGG OsU6b 3/5

Os 20 Os06g0275000 AACGTGTTCGACCAGGAGGTTGG OsU3 16/18

Os06g0275000 GGGTATAGTACCAGACAGCACGG OsU6a 17/19

Sum 46 targets 280/328 = 85.4%

At 1 At05g55580 ACAAAGAGAGGTGATTCCGACGG AtU3b 6/14

GATGAGTTCGAGGAAGTATGTGG AtU6-29 3/9

At 2

At 3

At 4

At1g56650

At1g03180

At1g16210

AAAACCTAAGTACATGTATTTGG

CCATGGAGGGTTCGTCCAAAGGG

GAAACTTCAATTTGCAGCTTTGG

ATCTCGTAAGAACGCCGCGGAGG

AtU3d

AtU6-29

AtU6-1

AtU3b

2/9

3/6

3/5

25/75

6 targets 42/118 = 35.6%

17

Supplementary Table 5. Targeted genomic mutations in T0 plants of rice and T1 plants of

Arabidopsis. Os 1-1~Os 21-20 are individual rice T0 plants with the constructs Os 1~Os 21, and At

1-1~At 4-74 are individual Arabidopsis T1 with the constructs At 1~At 4, as shown in

Supplementary Table 3. Varied nucleotides are highlighted in red.

Gene: OsFTL1;

Target sequence: GTCGGCCGGCAGCCGGATGA CGG; GC=75%; promoter: U6a

Heterozygous: 0; Biallelic: 3; Homozygous: 0; WT: 0; Total: 3

Os 1-1(1) GTCGGCCG-----------ACGG

GTCGGCCGGCAGC----TGACGG

biallelic

Os 1-2(1) CGTCGTCGGCCGGCAGCCGGA-GACGGGCAGTAGTTGTCCGGC

CGTCCGA---------------------------------GGC

CGTCGTCGGCCGGCAGCCGGATGACGGGCAGTAGTTGTCCGGC

biallelic

Os 1-3(1) GTCGGCCGGCAGCCG---TGACGG

GTCGGCCGGCAGCCGGACTGACGG

biallelic

Gene: OsFTL4;

Target sequence: GTCACGAGGTAGGGATCCTTTGG; GC=55%; promoter: U6b

Heterozygous: 0; Biallelic: 1; Homozygous: 2; WT: ; Total: 3

Os 1-1(2) GTCACGAGGTAGGGATC-TTTGG homozygous

Os 1-2(2) GTCACGAGGTAGGGATC-TTTGG homozygous

Os 1-3(2) GTCACGAGGTAGGGATC-TTTGG

GTCA---------------------TTGAGCC

biallelic

Gene: OsFTL5;

Target sequence: GACGCGCGGCTTGCCGGCGACGG; GC=80%; promoter: U6c


Os 1-1(3) GACGCGCGGCTTGCCGGACGACGG homozygous

Os 1-2(3) GACGCGCGGCTTGCCGGACGACGG homozygous

Os 1-3(3) GACGCGCGGCTTGCCGGGCGACGG

GACGCGCGGCTTGCCGGTCGACGG

biallelic

Gene: OsFTL6;

Target sequence: ATCTTCACTAGCCATGTCAA GGG; GC = 40%; promoter: U3


Os 1-1(4) ATCTTCACTAGCCATGTACAAGGG homozygous

Os 1-2(4) ATCTTCACTAGCCA---------ACCC

ATCTTCACTAGCC--GTCAAGGGACCC

biallelic

Os 1-3(4) ATCTTCACTAGCCA--TCAAGGG

ATCTTCACTAGCC----CAAGGG

biallelic

Gene: OsFTL9;

Target sequence: TATTGGTGGCACCGACCTGA GGG; GC = 55%; promoter :U6c


Os 1-1(5) TATTGGTGGCACCGACCTTGAGGG homozygous

Os 1-2(5) TATTGGTGGCACCGACCTTGAGGG homozygous

Os 1-3(5) TATTGGTGGCACCGACCATGAGGG

TATTGGTGGCACCGACCTTGAGGG

biallelic

Gene: OsFTL10;

Target sequence: GGCTTGCTTACAACTGCAGATGG; GC = 50%; promoter: U6a


Os 1-1(6) GGCTTGCTTACAAC--CAGATGG homozygous

Os 1-2(6) GGCTTGCTTACA------GATGG

GGCTTGCTT-----------TGG

biallelic

Os 1-3(6) GGCTTGCTTACAA---CAGATGG biallelic

18

GGCTTGCTTACAA-----GATGG

Gene: OsFTL11;

Target sequence: TCATGTCGTCGGCGATATCGTGG; GC= 55%; promoter: U6b


Os 1-1(7) TCATGTCGTCGGCGATATCGTGG WT



Gene: OsFTL13;

Target sequence: CGGTGCTGATGAAGGGATCCAGG; GC = 60%; promoter: U3


Os 1-1(8) ATACA-------------------------------------GG

ATACATGACGGTGAGATCGACGGTGCTGATGAAGGGATTCCAGG

biallelic

Os 1-2(8) CGGTGCTGATGAAGGG--TCCAGG

CGGTGCTGATGAAGGGATTCCAGG

biallelic

Os 1-3(8) CGGTGCTGATGAAGGGATTCCAGG

CGGTGCTGATGAAGGGAATCCAGG

biallelic

Gene:OsFTL7;

Target sequence: TCGGCGCGTCGGCGCTGCTGAGG; GC = 80%; Promoter: U6a


Os 2-1(1) TCGGCGCGTCGGCGC-CCTGAGG

TCGGCGCGTCGGCGC-GCTGAGG

biallelic

Os 2-2(1) TCGGCGCGTCGGCGC-CCTGAGG

TCGGCGCGTCGGCGC-TCTGAGG

biallelic

Os 2-4(1) TCGGCGCGTCGGCGCTGGCTGAGG

TCGGCGCGTCGGCGCTGTCTGAGG

biallelic

Os 2-5(1) TCGGCGCGTCGGCGCT--CTGAGG

TCGGCGCGTCGGCGCTGCCTGAGG

biallelic

Os 2-6(1) TCGGCGCGTCGGCGCT-CTGAGG

TCGGCGCGTCGGCGCT--TGAGG

biallelic

Os 2-7(1) TCGGCGCGTCGGCGCTGACTGAGG

TCGGCGCGTCGGCGCT—CTGAGG

biallelic


TCGGCGCGTCGGCGCTG----AGG

biallelic

Os 2-9(1) TCGGCGCGTCGGCGCTGTCTGAGG homozygous

Os 2-10(1) TCGGCGCGTCGGCGCTGCCTGAGG

TCGGCGCGTCGGCGCTGGCTGAGG

biallelic

Os 2-11(1) TCGGCGCGTCGGCGCTGTTCTGAGG

TCGGCGCGTCGGCGCTG-----AGG

biallelic


TCGGCGCGTCGGCGCTGTCTGAGG

biallelic

Os 2-13(1) TCGGCGCGTCGGCGCT-CTGAGG homozygous


TCGGCGCGTCGGCGCT(36 bp deletion)CGGGTC

biallelic


TCGGCGCGTCGGCGCTGGCTGAGG

biallelic

Os 2-17(1) TCGGCGCGTCGGCGCT--CTGAGG

TCGGCGCGTCGGCGCTGCCTGAGG

biallelic

Os 2-18(1) TCGGCGCGTCGGCGCCTGCTGAGG

TCGGCGCGTCGGCGC-TGT—AGG

biallelic

Os 2-19(1) TCGGCGCGTCGGCGCTGTCTGAGG homozygous

Os 2-20(1) TCGGCGCGTCGGCGCTGTCTGAGG

TCGGCGCGTCGGCGCT—CTGAGG

biallelic


TCGGCGCGTCGGCGCTG----AGG

biallelic

19

Os 2-24(1) TCGGCGCGTCGGCGCC-CTGAGG

TCGGCGCGTCGGCGCG-CTGAGG

biallelic


TCGGCGCGTCGGCGCTGACTGAGG

biallelic


TCGGCGCGTCGGCGCTGACTGAGG

biallelic

Gene: OsFTL8;

Target sequence: CGGCGAACAGCCTGGTGCTGGGG; GC = 70%; promoter: U6b


Os 2-1(2) CGGCGAACAGCCTGGTGTCTGGGG homozygous

Os 2-2(2) CGGCGAACAGCCTGGTGACTGGGG homozygous

Os 2-3(2) CGGCGAACAGCCTGGTGGCTGGGG

CGGCGAACAGCCTGGTGTCTGGGG

biallelic

Os 2-4(2) CGGCGAACAGCCTGGTGGCTGGGG


biallelic


Os 2-6(2) CGGCGAACAGCCTGGTGTCTGGGG

CGGCGAACAGCCTGGTGACTGGGG

biallelic

Os 2-7(2) CGGCGAACAGCCTGGTGGCTGGGG homozygous



biallelic

Os 2-9(2) CGGCGAACAGCCTGGT----GGGG


biallelic



biallelic


Os 2-12(2) CGGCGAACAGCCTGGTGCCTGGGG


biallelic


Os 2-14(2) CGGCGAACAGCCTGGTGACTGGGG

CGGCGAACAGCCTGGTG------G

biallelic



biallelic




biallelic


CGGCGAACAGCCTGGTGGCTGGGG

biallelic


CGGCGAACAGCCTGGTGGCTGGGG

biallelic



biallelic

Os 2-24(2) CGGCGAACAGCCTGGTGTCTGGGG homozygous


Gene: OsFTL12;

Target sequence: TGGCAAGGAGTTCCGTTCCTCGG; GC = 55%; promoter: U6c


Os 2-1(3) TGGCAAGGAGTTCCGT--CCTCGG

TGGCAAGGAGTTCCGTTACCTCGG

biallelic

Os 2-2(3) TGGCAAGGAGTTCCGTT-CCTCGG


heterozygous

Os 2-3(3) TGGCAAGGAGTTCCGTTC-TCGG

TGGCAAGGAGTTCCGTTCCTCGG

heterozygous

Os 2-4(3) TGGCAAGGAGTTCCGTT--CTCGG


biallelic

20

Os 2-5(3) TGGCAAGGAGTTCCGTTCCCTCGG homozygous

Os 2-6(3) TGGCAAGGAGTTCCG--------G

TGGCAAGGAGTTCCGTTTCCTCGG

biallelic

Os 2-7(3) TGGCAAGGAGTTCCGTTTCCTCGG

TGGCAAGGAGTTCC-----CTCGG

biallelic

Os 2-9(3) TGGCAAGGAG--------------------------ACCT

TGGCA--------------------GTCTCGATGAAACCT

biallelic

Os 2-10(3) TGGCAAGGAGTTCCGTTACCTCGG

TGGCAAGGAGTTCCGTTCCCTCGG

biallelic

Os 2-11(3) TGGCAAGGAGTTCCGT-CCTCGG homozygous


TGGCAAGGAGTTCCGTT-CCTCGG

heterozygous


TG---------------------------------AAAC

biallelic

Os 2-14(3) TGGCAAGGAGTTCCGTTTCCTCGG homozygous

Os 2-15(3) TGGCAAGGAGTTCCGTTCCCTCGG


biallelic

Os 2-17(3) TGGCAAGGAGTTCCGTTCCCTCGG homozygous



biallelic


TGGCAAGGAGTTCCGTT-CCTCGG

heterozygous


TGGCAAG--------TT-CCTCGG

biallelic

Os 2-22(3) TGGCAAGGAGTTCCGTTTCCTCGG homozygous


TGGCAAGGAGTTCCGT--CCTCGG

biallelic

Os 2-24(3) TGGCAAGGAGTTCCGT--CCTCGG


biallelic

Os 2-25(3) TGGCAAGGAGTTCCGTTCCCTCGG

TGGCAAGGAGTTCCGTTTCCTCGG

biallelic

Gene：OsGSTU;

Target sequence: GGCGGCCGCTCTTCCGCGGGTGG; GC = 85%; promoter: U6a


Os 3-1(1) GGCGGCCGCTCTTCCGCGGGTGG

GGCGGGCAGGA----------GG

heterozygous

Os 3-2(1) GGCGGCCGCTCTTCCGCTGGGTGG homozygous

Os 3-3(1) GGCGGCCGCTCTTCCGCTGGGTGG homozygous

Os 3-4(1) GGCGGCCGC--------GGGTGGAGCGCCTCCT

GGCGGCCGCT-----------------CCTCCT

biallelic

Os 3-5(1) GGCGGCCGCTCTTCC-------GG

GGCGGCCGCTCTTCCGCTGGGTGG

biallelic

Gene：OsMRP15;

Target sequence: TGCTAAAACTGAAACTAGTAAGG; GC = 30%; promoter: U3


Os 3-2(2) TGCTAAAACTGAAAC-AGTAAGG homozygous

Os 3-3(2) TGCTAAAACTGAAAC--GTAAGG

TGCTAAAACTGAAACTAGTAAGG

heterozygous

Os 3-4(2) TGCTAAAACTGAAACTAGTAAGG

TGCTAAAACTGAAACTA---AGG

heterozygous

Gene：OsAnp;

Target sequence: GGAGGAGGACGCGGCCGCCGTGG; GC = 85%; promoter: U6c


Os 3-1(3) GGAGGAGGACGCGGCCGACCGTGG biallelic

21

GGAGGAGGACGCGGCC--CCGTGG

Os 3-2(3) GGAGGAGGACGCGGCCGACCGTGG

GGAGGAGGACGCGGCC--CCGTGG

biallelic

Os 3-3(3) GGAGGAGGAC-----------------------G

GGAGGAGGACGCGGCC-CCGTGGAGTCGGCCGGG

biallelic

Os 3-4(3) GGAGGAGGAC----------------------GG homozygous

Gene：Waxy

Target sequence: TGTGTGCTTACAGCCATGGCAGG; GC = 55%; promoter: U6a;


Os 4-1(1) TGTGTGCTTACAGCCATTGGCAGG

TGTGTGCTTACAGCCAT-GGCAGG

heterozygous

Os 4-3(1) TGTGTGCTTACAGCCATTGGCAGG homozygous

Gene：Waxy

Target sequence: GAGCCTCGAGTGCTGCCTGCAGG; GC = 70%; promoter: U3;


Os 4-1(2) GAGCCTCGAGTGCTGCCTTGCAGG

GAGCCTCGAGTGCTGCC--GCAGG

biallelic

Os 4-2(2) GAGCCTCGAGTGCTGCCTTGCAGG homozygous

Gene：Waxy

Target sequence: GTCTGAATCTTTTTCACTGCAGG; GC = 40%; promoter: U6b;


Os 4-3(3) GTCTGAATCTTTTTCACTTGCAGG

GTCTGAATCTTTTTCAC-TGCAGG

heterozygous

Gene: Os05g0591600

Target sequence: CCAGGTATTATAGCCGATCCTCT; GC = 45%; promoter: U3;


Os 5-1(1) CCAGGTATTATAGCCGATCCTCT

CCAGGT-----AGCCGATCCTCT

heterozygous

Os 5-2(1) CCAGGT----ATAGCCGATCCTCT

CCAGGTAATTATAGCCGATCCTCT

Biallelic

Os 5-3(1) CCAGGTAATTATAGCCGATCCTCT

CCAGGTATTTATAGCCGATCCTCT

biallelic

Os 5-4(1) CCAGGTATTTATAGCCGATCCTCT

CCAGGTAATTATAGCCGATCCTCT

Biallelic

Gene: Os03g0126800;

Target sequence: CCCAACTGCAAATACAAGGTGCA; GC = 40%; promoter: U6a


Os 6-1(1) CCCAAC(deletion between two sites)TACAAGG homozygous

Os 6-2(1) CCCAACT(deletion between two sites)TGCAAATACAAGG homozygous


Os 6-4(1) CCCAACT(deletion between two sites)AGGTGC

CCCTACAT(deletion between two sites)GCAAAT

biallelic

Os 6-5(1) CCCAACTGCAAAT(deletion between two sites)TACAAG

CCCAACTGC (deletion between two sites)ACAAGG

biallelic

Os 6-6(1) CCCAAC-TACAAA(deletion between two sites)TACAAG

CCCAACTTGC(deletion between two sites)GGTGCA

biallelic

Os 6-7(1) CCCAAC-TACAAA(deletion between two sites)TACAAG

CCCAACTTGC(deletion between two sites)GGTGCA

biallelic

Os 6-8(1) CCCAACT(deletion between two sites)TGCAAA

CCCAA(deletion between two sites) ATACAA

biallelic

Os 6-9(1) CCCAACT(deletion between two sites)TGCAAAT biallelic

22

CCCAAC(deletion between two sites)CAAATA


CCCAACAGC-AATACAAGGTGCA

biallelic

Os 6-11(1) CCCAACTTGCAAATACAAGGTGCA homozygous


CCCAACTTGCAAATACAAGGTGCA

biallelic

Os 6-13(1) CCCAACTTGCAAATACAAGGTGCA

CCCAAC-T（deletion between two sites）GCAAAT

biallelic

Gene: Os03g0126800;

Target sequence: AAATCCCAACTGCAAATACAAGG;GC = 35%; promoter: U3


Os 6-1(2) CCCAAC(deletion between two sites)TACAAGG homozygous



Os 6-4(2) CCCAACT(deletion between two sites)AGG

CCTACAT(deletion between two sites)GCAAATACAAGG

biallelic

Os 6-5(2) TGCAAA(deletion between two sites)TACAAGG

AACTGC(deletion between two sites)ACAAGG

biallelic

Os 6-6(2) CAAA(deletion between two sites)CAAATACAAGG

CCAACTTGC(deletion between two sites)GG

biallelic

Os 6-7(2) ATACAAA(deletion between two sites)CAAATACAAGG

ACTTGC(deletion between two sites)GG

biallelic

Os 6-8(2) CCCAACT(deletion between two sites)TGCAAATACAAGG

TCCCAA(deletion between two sites)ATACAAGG

biallelic

Os 6-9(2) CCCAACT(deletion between two sites)GCAAATTACAAGG

CCCAAC(deletion between two sites)CAAAT-ACAAGG

biallelic

Os 6-10(2) CCCAACT(deletion between two sites)TGCAAATACAAGG

AAATCCCAACTGCAAATACAAGG

heterozygous

Os 6-11(2) AAATCCCAACTGCAAATAAC-AGG

AAATCCC(36 bp deletion)ATACAT

biallelic

Os 6-12(2) AAATCCCAACTGTCAAATACAAGG

CCCAACT(deletion between two sites)TGCAAATACAAGG

biallelic

Os 6-13(2) AAATCCCAACTGCAAATACAAGG

CCCAACT(deletion between two sites)GCAAATACAAGG

heterozygous

Gene: Os07g0409500;

Target sequence: GCGAGCGAGAGTCATGGAGGCGG; GC = 60%; promoter: U6a


Os 7-1(1) GCGAGCGAGAGTCATGG(deletion between two

sites)GACAAGG

homozygous

Os 7-2(1) GCGAGCGAGAGTCA---AGGCGG homozygous

Os 7-3(1) GCGAGCGAGAGTCATGGTAGGCGG

GCGAGCGAGAGT-------GGCGG

biallelic

Os 7-4(1) GCGAGCGAGAGTC(30 bp deletion)AAGGCC

GCGAGCGAGAGTCA---GGCGG

biallelic

Os 7-6(1) GCGAGCG(25 bp deletion)GGGAGG homozygous

Gene: Os07g0409500;

Target sequence: GCACGCCAAGGTCGACGACAAGG; GC = 65%; promoter: U6a


Os 7-1(2) TCATGG (deletion between two sites)GACAAGG homozygous

Os 7-2(2) GCACGCCAAGGTCGAC-ACAAGG homozygous

Os 7-3(2) GCACGCCAAGGTCGACGAACAAGG homozygous

Os 7-6(2) GCACGCCAAGGTCGA--ACAAGG homozygous

Gene: Os07g0625500;

23

Target sequence: GCGAGATCGAGCGATGGCGA CGG; GC = 65%; promoter: U6a


Os 8-2I(1) GCGAGATCGAGCGATGGGCGACGG

GCGAGATCGAGCGATGGTCGACGG

biallelic

Os 8-3(1) GCG(24 bp deletion)CGGAGC

GCGAGATCGAGC-----CGACGG

biallelic

Os 8-4(1) GCGAGATCGAGCGATGGACGACGG

GCGAGATCGAGCGATGGGCGACGG

biallelic

Gene: Os07g0625500;

Target sequence: GGCGCTCGCCAAGTTCGACA GGG; GC = 65%; promoter: U6a


Os 8-2(2) GGCGCTCGCCAAGTTCGTACAGGG

GGCGCTCGCCAAGTTCGGACAGGG

biallelic

Os 8-4(2) GGCGCTCGCCAAGTTCGAACAGGG homozygous

Os 8-5(2) GGCGCTCGC---------ACAGGG

GGCGCTCGCCAAGTTCGGACAGGG

biallelic

Gene: Os07g0261200;

Target sequence: ATTTATCCGTTCATGTCGAT GGG; GC = 35%; promoter: U3


Os 9-11(1) ATTTATCCGTTCATGTCAGATGGG

ATTTATCCGTTCATGTCTGATGGG

biallelic

Os 9-15(1) ATTTATCCGTTCATGT-GATGGG

ATTTATCCGTTCATGTCGATGGG

heterozygous

Gene: Os07g0261200;

Target sequence: GATACTCACGATGGTGGCGCTGG; GC = 60%; promoter: U6a;


Os 9-1(2) GATACTCACGATGGTGGACGCTGG

GATACTCACGATGGTGGTCGCTGG

biallelic

Os 9-2(2) GATACTCACGATGGT---CGCTGG

GATACTCACGATGGTGGACGCTGG

biallelic

Os 9-3(2) GATACTCACGATGGT---CGCTGG


biallelic


GATACTCACGATGGTGGGCGCTGG

biallelic



biallelic



biallelic



biallelic

Os 9-8(2) GATACTCACGATGGTGGGCGCTGG

GATACTCACGATGGTGGGCGCTGG

homozygous

Os 9-9(2) GATACTCACGATGGTG--CGCTGG

GATACTCACGATGGTGGWCGCTGG

chimeric



homozygous

Os 9-12(2) GATACTCACGATCAGGGGTGTTGG


biallelic



homozygous

Os 9-14(2) GATACTCACGATGGTGG-(11 bp deletion)CTCGGC

GATACTCACGATGGTGGACGCTGGCCGCGC

biallelic

Os 9-16(2) GATACTCACGATGGTGGGCGCTGG biallelic

24

GATACTCACGATGGTCAATCTTATAT(23 bp deletion)GCCGCC



biallelic



biallelic



biallelic

Os 9-21(2) GATACTCACGATGGTGCGCGCTGG

GATACTCACGATGGTG-G-ACTGG

biallelic

Os 9-22(2) GATACTCACGATGGTGG-CGCTGG

GATACTCACGATGGTGGCCGCTGG

heterozygous

Gene: Os05g0543000;

Target sequence: AACCTGATCACCAACCCACTCGG; GC = 50%; promoter: U3


Os 10-1(1) AACCTGATCACCAACCCACTCGG

AACCTGATCACCA----ACTCGG

heterozygous

Gene:Os11g0549665;

Target sequence: ATCTGGTGGGGCTGATCGGGGGG; GC = 65%; promoter: U3


Os 10-1(2) ATCTGGTGGGGCTG---GGGGGG ATCTGGTGGGGC---------GG biallelic

Gene: Os10g0548600;

Target sequence: GTGGTGGCAGCGCTGGTCGGCGG; GC = 75%; promoter: U6a


Os 10-1(3) GTGGTGGCAGCGCTGGTACGGCGG

GTGGTGGCAGCGCTGGTTCGGCGG

biallelic

Gene: Os05g0312500;

Target sequence: AAGATCCATGAGTTCAAGAA GGG; GC = 35%; promoter: U3


Os 11-1(1) AAGATCCATGAGTTCAAAGAAGGG

AAGATCCATGAGTTCA--GAAGGG

biallelic

Os 11-2(1) AAGATCCATGAGT---AGAAGGG

AAGATCCATGAGT----GAAGGG

biallelic

Os 11-3(1) AAGATCCATGAGTTCAATGAAGGG

AAGATCCATGAGTTC---GAAGGG

biallelic


AAGATCCATGAG-------AAGGG

biallelic


AAGATCCATGAG-------AAGGG

biallelic

Gene: Os04g0668400;

Target sequence: GCGGAAGCCTCGACAGTCACCGG; GC = 65%; promoter: U6a


Os 11-1(2) GCGGAAGCCTCGACAGTTCACCGG

GCGGAAGCCTCGACAGTACACCGG

biallelic

Os 11-2(2) GCGGAAGCCTCGACAGTACACCGG

GCGGAAGCCTCGACAGTTCACCGG

biallelic

Os 11-3(2) GCGGAAGCCTCGAC----CACCGG


biallelic

Os 11-4(2) GCGGAAGCCTCGAC----CACCGG


biallelic

Os 11-5(2) GCGGAAGCCTCGACAGTTCACCGG homozygous

Gene: Os07g0411300;

25

Target sequence: GTCGTCCATGGCCCCCAACCCGG; GC = 70%; promoter: U6a


Os 12-1(1) GTCGTCCATGGCCCC--ACCCGG

GTCGTCCATGGCCCC-----CGG

biallelic

Os 12-2(1) GTCGTCCATGGCC-------CGGGCAAGGCCAC

GTCGTCCATGGCC---------------GCCAC

GTCGTCCATGGCCCCCAACCCGGGCAAGGCCAC (Ref)

biallelic

Os 12-3(1) GTCGTCCATGGCCA----CCCGGGCAAGGCCAC

GTCGTCCATGGCCA-----------------AC

GTCGTCCATGGCCCCCAACCCGGGCAAGGCCAC (Ref)

biallelic

Os 12-6(1) GTCGTCCATGGCC----ACCCGG homozygous

Os 12-7(1) GTCGTCCATGGCC----ACCCGG

GTCGTCCATGGCCCCC--CCCGG

biallelic

Os 12-8(1) GTCGTCCATGGCCCCC------CGG

GTCGTCCATGGCCCCCACCACCCGG

biallelic

Os 12-10(1) GTCGTCCATGGCC-----CCCGG

GTCGTCCATGGC------CCCGG

biallelic

Os 12-11(1) GTCGTCCATGGCCCC---ACCCGG

GTCGTCCATGGCCCCCACACCCGG

biallelic

Os 12-12(1) GTCGTCCATGGCCC----CCCGG

GTCGTCCATTGGCC--------G

biallelic

Gene: Os10g0484800;

Target sequence: ACCTTGGATTCGGCGATGCCAGG; GC = 60%; promoter: U3


Os 12-1(2) GC----------------------------GCCAGG

GCGAAGATGGCTGACCTTGGATTCGGCGATGCCAGG (Ref)

homozygous

Os 12-2(2) ACCTTGGATTCGGCG-TGCCAGG

ACCTTGGATTCGGCGTTGCCAGG

biallelic

Os 12-8(2) ACCT------------TGCCAGG homozygous

Os 12-9(2) ACCTTGGATTCGGCGATTGCCAGG homozygous



Os 12-12(2) ACCTTGGATTCGGCGATTGCCAGGAGTG

ACCT----------------------TG

biallelic

Os 12-14(2) ACCTTGGATTCGGCG-TGCCAGG

ACCTTGGATTCGGCG (31 bp deletion) ATGCTCC

biallelic

Gene: Os04g0595000;

Target sequence: CCCTCTGCAGAGGAGGATCGAGG; GC = 60%; promoter: U6b


Os 13-1(1) CCCTCATGCAGAGGAGGATCGAGG

CCCTCATGCAGAGGAGGATCGAGG

homozygous

Gene: Os12g0242700;

Target sequence: CCTGACTTGGCATAGTTCACAAT; GC = 40%; promoter: U3


Os 14-1(1) CCTGACATTGGCATAGTTCACAAT

CCTGAC----GCATAGTTCACAAT

biallelic

Os 14-2(1) CCTGACATTGGCATAGTTCACAAT

CCTGAC----GCATAGTTCACAAT

biallelic

Os 14-3(1) CCTGACTTTGGCATAGTTCACAAT homozygous

Os 14-4(1) CCTGAC--GGCATAGTTCACAAT homozygous

Os 14-5(1) CCTGACT-GGCATAGTTCACAAT homozygous

Os 14-6(1) CCTGACTTTGGCATAGTTCACAAT homozygous

Os 14-7(1) CCTGAC---GCATAGTTCACAAT homozygous

Os 14-8(1) CCTGACTTTGGCATAGTTCACAAT biallelic

26

CCTGAC-T--GCATACTTCACAAT

Os 14-9(1) CCTGAC--GGCATAGTTCACAAT homozygous

Os 14-10(1) CCTGACT--GCATAGTTCACAAT

CCTGAC---GCATATAGTTCAAT

biallelic

Gene: Os03g0216800;

Target sequence: GGAGGCTCCATCGAATCGATCGG; GC = 55%; promoter: U6b


Os 15-1(1) GGAGGCTCCATCGAAT--GATCGG

GGAGGCTCCATCGAATCAGATCGG

biallelic

Os 15-2(1) GGAGGCTCCATCG----GATCGG

GGAGGCTCCATCGAA--GATCGG

biallelic

Os 15-3(1) GGAGGCTCCATCGAATCAGATCGG homozygous

Os 15-4(1) GGAGGCTCCATCGAA--GATCGG homozygous

Os 15-6(1) GGAGGCTCCATCGAATCAGATCGG homozygous

Os 15-7(1) GGAGGCTCCATCGAATCCGATCGG

GGAGGCTCCATCGAATCAGATCGG

biallelic

Os 15-8(1) GGAGGCTCCATCGAATCTGATCGG homozygous

Gene: Os02g0459600;

Target sequence: GGCTATTGGATCGTGCACCAAGG; GC = 55%; promoter: U6b;


Os 16-1(1) GGCTATTGGATCGTGCATCCAAGG homozygous

Os 16-2(1) GGCTATTGGATC--------AAGG homozygous

Gene: Os01g0891000;

Target sequence: TCACCTTGCAAACATGGCGCAGG; GC = 55%; promoter: U6a;


Os 16-1(2) TCACCTTGCAAACATGGTCGCAGG homozygous

Os 16-2(2) TCACCTTGCAAACATGGGCGCAGG homozygous

Gene: Os10g0413900;

Target sequence: CCTCGCCGCCGTACGTGTAGCGG; GC = 70%; promoter: U6a;


Os 17-1(1) CCTCGCCGCCTACGTGTTAGCGG

CCTCGCCGCCTACGTG (86 bp deletion between two targets)

CACCGG

biallelic

Os 17-2(1) CCTCGCCGCCGTACGTGTAGCGGAGCGACTCCCTCTCTCCC

C--------------------------------TCTCTCCC

heterozygous

Os 17-4(1) CCTCGCCGCCGTACGTGTTAGCGG homozygous

Os 17-5(1) CCTCGCCGCCGTACGTGTTAGCGG

CCTCGCCGCCGTACGTGCTAGCGG

biallelic

Os 17-6(1) CCTCGCCGCCGTACGTGTAGCGGAGCGACTCCC

CCTCGCCGC-----------------GTCTCCC

heterozygous

Os 17-8(1) CCTCGCCGCCGTAC----TAGCGG

CCTCGCCGCCGTACGTGCTAGCGG

biallelic

Os 17-9(1) CCTCGCCGCCGTACGTGT-------GCGACT

CCTCGCCGCCGTACGTGTTAGCGGAGCGACT

biallelic

Os 17-10(1) CCTCGCCGCCGTA----TAGCGG homozygous

Gene: Os10g0413900;

Target sequence: CCGGTGCCGCCATCCCCTCGCCC; GC = 80%; promoter: U6b


Os 17-1(2) CCGGTGACCGCCATCCCCTCGCCC

CCGGTG (86 bp deletion between two targets)CACGTA

biallelic

Os 17-2(2) CCGGTGCCCGCCATCCCCTCGCCC

CCGGTGTCCGCCATCCCCTCGCCC

biallelic

27

Os 17-3(2) CCGGTG--GCCATCCCCTCGCCC homozygous

Os 17-4(2) ACGC (33 bp deletion)CCGCCATCCCCTCGCCC homozygous

Os 17-5(2) GCCTTCCGGTGC-----CCGCCATCCCCTCGCCC

GCC---------------CGCCATCCCCTCGCCC

GCCTTCCGGTGCCGGTGCCGCCATCCCCTCGCCC (Ref)

biallelic

Os 17-6(2) CCGGTGTCCGCCATCCCCTCGCCC

CCGGTG--CGCCATCCCCTCGCCC

biallelic

Os 17-8(2) CCGGT(46 bp deletion)CCCGGAGGTG

CCGGTGTCCGCCATCCCCTCGCCCCGTCGATCGATCGACCGACCGACCG

GTCCCGGAGGTG

CCGGTG-CCGCCATCCCCTCGCCCCGTCGATCGATCGACCGACCGACCG

GTCCCGGAGGTG (Ref)

biallelic

Os 17-9(2) CCGGTGTCCGCCATCCCCTCGCCC

CCGGTGACCGCCATCCCCTCGCCC

bi-alleic

Os 17-10(2) CCGGTGACCGCCATCCCCTCGCCC homozygous

Gene: Os06g0142000;

Target sequence: TTCATGCTCAGGCATCGCTATGG; GC = 50%; promoter: U3;


Os 18-1(1) TTCATGCTCAGGCATCGGCTATGG

--------------TCACCTATGG

biallelic

Os 18-2(1) TTCATGCTCAGGCATCGCCTATGG

TTCATGCTCAGGCATCGACTATGG

biallelic

Os 18-4(1) TTCATGCTCAGGCATCGCCTATGG

TTCATGCTCAGGCATCGTCTATGG

biallelic

Gene: Os06g0142100;

Target sequence: CATGGGGAACTTGGACTTGGCGG; GC = 55%;Promoter: U6a;


Os 18-1(2) CATGGGGAACTTGGACTTTGGCGG

CATGGGGAA---------TGGCGG

biallelic

Os 18-2(2) CATGGGGAACTTGGAC-TGGCGG homozygous

Gene: Os03g0247300

Target sequence: CGCAAGACAGGGCACACCGGAGG; GC = 70%; promoter: U6b


Os 19-1(1) CGCAAGACAGGGCACACTCGGAGG

CGCAAGACAGGGCACAC--GGAGG

biallelic

Os 19-2(1) CGCAAGACAGGGCACACACGGAGG

CGCAAGACAGGGCACACACGGAGG

homozygous

Os 19-3(1) CGCAAGACAGGGCACACACGGAGG

CGCAAGACAGGGCACACACGGAGG

homozygous

Gene: Os06g0275000

Target sequence: AACGTGTTCGACCAGGAGGTTGG; GC = 55%; promoter: U3


Os 20-1(1) AACGTGTTCGACCAGGATGGTTGG homozygous

Os 20-2(1) AACGTGTTCGACCAGGGTGGTTGG

AACGTGTTCGACCAGG-AGGTTGG

heterozygous

Os 20-3(1) AACGTGTTCGAC------------GTTGG

AACGTGTTCGACCAG--GGTTGGAGTTGG

biallelic

Os 20-4(1) AACGTGTT--------T-GGTTGG

AACGTGTTCGACCAGGATGGTTGG

biallelic

Os 20-5(1) AACGTGTTCGACCAGG----GGTTGG

AACGTGTTCGACCAGGATAAGGTTGG

biallelic

Os 20-7(1) AACGTGTTCGACC-----GGTTGG biallelic

28


Os 20-9(1) AACGTGTTCGACCAGGAAGGTTGG

AACGTGTTCGACCAGGAGGGTTGG

biallelic

Os 20-10(1) AACGTGTTCGACCAGG---GTTGG


biallelic



biallelic

Os 20-12(1) AACGTGTTCGACCAGG-----TTGG

AACGTGTTCGACCAGGAAAGGTTGG

biallelic

Os 20-13(1) AACGTGTTCGAC（31 bp deletion）GAGGAGGAGA

AACGTGTTCGACC-----GTTGG

biallelic

Os 20-14(1) AACGTGTTCGACCAGG--GGTTGG


biallelic


AACGTGTTCGACCAGGAGGGTTGG

biallelic



biallelic

Os 20-19(1) AACGTGTTCGA（25bp deletion）GGAGGAGGAGG

AACGTGTTCGACCAGTTAGGTTGG

biallelic

Os 20-20(1) AACGTGTTCGACCAGG（22 bp deletion）AGGAG

AACGTGTTCGACCAGG---TTGG

biallelic

Gene: Os06g0275000

Target sequence: CCGTGCTGTCTGGTACTATACCC; GC = 50%; promoter: U6a


Os 20-1(2) CCGTGC----CTGGTACTATACCC

CCGTGCTTGTCTGGTACTATACCC

biallelic

Os 20-2(2) CCGTGC-GTCTGGTACTATACCC

CCGTGCT-----GTACTATACCC

biallelic

Os 20-3(2) CCGTGC--TCTGGTACTATACCC

CCGTGC-GTCTGGTACTATACCC

biallelic

Os 20-4(2) CCGTGCT---CTGGTACTATACCC


biallelic

Os 20-5(2) CCGTGCT---TGGTACTATACCC

CCGTGCT----GGTACTATACCC

biallelic

Os 20-7(2) CTAT----------------TGGTACTATACCC

ATATCTATCACCGTGCTGTCTGGTACTATACCC

heterzygous

Os 20-8(2) CCGTGC-GTCTGGTACTATACCC homozygous

Os 20-9(2) CCGTGC------GGTACTATACCC


biallelic

Os 20-10(2) C---------TGGTACTATACCC

CCGTGC-GTCTGGTACTATACCC

biallelic

Os 20-11(2) CATAT(17 bp deletion)GGTACTATCCC

ATATCTATCACCGT-CTGTCTGGTACTATCCC

biallelic

Os 20-12(2) CCGTGCT-----------ATACCC


biallelic

Os 20-13(2) CCGTGC--GTCTGGTACTATACCC


biallelic

Os 20-14(2) CCGTGC---CTGGTACTATACCC

CCGTG-TGTCTGGTACTATACCC

bialleic

Os 20-16(2) CCGTGC------GGTACTATACC

CCGTGCTTGTCTGGTACTATACC

biallelic

Os 20-17(2) CCGTGC-----GGTACTATACCC

CCGTGCT-TCTGGTACTATACCC

biallelic

Os 20-18(2) CCGTGCT-----GGTACTATACCC


biallelic

Os 20-19(2) CCGTGCT-----GGTACTATACCC bialleic

29


Gene: At5g55580

Target sequence: ACAAAGAGAGGTGATTCCGA CGG; GC = 35%；Promoter: AtU3b

Heterozygous: 4; Biallelic: 2; Homozygous: 0; Chimaric:4; WT: 4; Total: 14

At 1-1(1) ACAAAGAGAGGTGATTACCGACGG

ACAAAGAGAGGTGATTCCCGACGG

biallelic

At 1-2(1) ACAAAGAGAGGTGA--CCGACGG

ACAAAGAGAGGTGATTCCGACGG

heterozygous

At 1-3(1) ACAAAGAGAGGTGAT-CCGACGG


heterozygous

At 1-4(1) ACAAAGAGAGGTGATT-CGACGG

ACAAAGAGAGGTGATTCGACGG

heterozygous

At 1-5(1) ACAAAGAGAGGTGAT-CCGACGG

ACAAAGAGAGGTGA--CCGACGG

biallelic

At 1-6(1) ACAAAGAGAGGTGATT-CGACGG


heterozygous

At 1-10(1) chimaric

At 1-11(1) chimaric

At 1-12(1) chimaric

At 1-13(1) chimaric

Gene: At5g55580

Target sequence: GATGAGTTCGAGGAAGTATGTGG; GC = 45%；Promoter: AtU6-29


At 1-3(2) GATGAGTTCGAGGAAGTTATGTGG

GATGAGTTCGAGGAAGT-ATGTGG

heterozygous

At 1-5(2) GATGAGTTCGAGGAAGTTATGTGG

GATGAGTTCGAGGAAGTAATGTGG

biallelic

At 1-6(2) GATGAGTTCGAGGAAG-ATGTGG

GATGAGTTCGAGGAAGTATGTGG

heterozygous

Gene: At5g55580

Target sequence: AAAACCTAAGTACATGTATTTGG; GC = 25%；Promoter: AtU3d


At 1-4(3) AAAACCTAAGTACATGTTTTTGG


heterozygous

At 1-8(3) AAAACCTAAGTACA-GTATTTGG


heterozygous

Gene: At1g56650

Target sequence: CCATGGAGGGTTCGTCCAAAGGG；Promoter: AtU6-29


At 2-1 CCATGGAAGGGTTCGTCCAAAGGG heterozygous

At 2-2

CCATGG-AGGGTTCGTCCAAAGGG

CCATGGAAGGGTTCGTCCAAAGGG

homozygous

At 2-3 CCATGGA---TTCGTCCAAAGGG homozygous

Gene: At1g03180

Target sequence: GAAACTTCAATTTGCAGCTTTGG；Promoter: AtU6-1

Heterozygous: 3 ; Biallelic: 0 ; Homozygous: 0; WT: 2 ; Total: 5

At 3-1 GAAACTTCAATTTGCAGCTTTGG

GAAACTTCAATTTGGACCTTTGG

heterozygous

At 3-2 GAAACTTCAATTTGCA-CTTTGG heterozygous

30


At 3-3 GAAACTTCAATTTGGAGCTTTGG


heterozygous

Gene: At1g16210

Target sequence: ATCTCGTAAGAACGCCGCGGAGG; GC = 60%；Promoter: AtU3b

Heterozygous: 15; Biallelic: 4; Chimeric：6；Homozygous: 0; WT: 50; Total: 75

At 4-1 ATCTCGTAAGAACGCC--CGGAGG

ATCTCGTAAGAACGCCGTCGGAGG

biallelic

At 4-2

At 4-4

GGCGGAGGTCGCCAAATCTCGTAAGAACGCCGCGGAGGCTGAGC

GGCGGAGG------------------------------CTGAGC

ATCTCGTAAGAACGCCG-CGGAGG

ATCTCGTAAGAACGCCGGCGGAGG

heterozygous

heterozygous

At 4-8

At 4-9

At 4-10

At 4-12

At 4-15

At 4-16

At 4-17

At 4-18

At 4-21

At 4-22

At 4-23

At 4-24

At 4-29

At 4-34

At 4-35

At 4-36

At 4-39

At 4-44

At 4-50


ATCTCGTAAGAACGCCGACGGAGG


ATCTCGTAAGAACGCCG---GAGG

ATCTCGTAAGAACGCCGACGGAGG(minor allele)

ATCTCGTAAGAACGCCGCGGAGG

ATCTCGTAAGAACGC--CGGAGG


ATCTCGTAAGAACGCCGCCGGAGG





GGCGGAGG------------------------------CTGAGC



ATCTCGTAAGAACGCCG---GAGG


ATCTCGTAAGAACG------GAGG









ATCTCGTAAGAACGCCGTCGGAGG(minor allele)







ATCTCGTAAGAACGCCGGCGGAGG(minor allele)



ATCTCGTAAGAA----G-CGGAGG



GGCGGAGG------------------------------CTGAGC




heterozygous

chimaric

(triallelic)

heterozygous

heterozygous

heterozygous

heterozygous

heterozygous

chimaric

(triallelic)

biallelic

biallelic

heterozygous

beterozygous

chimaric

(triallelic)

heterozygous

chimeric

chimaric

(triallelic)

heterozygous

heterozygous

biallelic

31

At 4-51

At 4-56

At 4-74



ATCTCGTAAGAACG------GAGG





chimaric

(triallelic)

heterozygous

heterozygous

Supplementary Table 6. Inheritance and genetic segregation of the edited sites in rice T1 lines. The

genotypes of the targeted sites in the T1 individuals were determined by sequencing.

T1 line Targeted

gene

T0 genotype (code) T1 genotype

(No. plants)

χ2

(1:2:1)

Os 2-10 FTL7 TCGGCGCGTCGGCGCTGCCTGAGG (107a)

TCGGCGCGTCGGCGCTGGCTGAGG (107b)

107a/107a (7)

107a/107b (18)

107b/107b (8)

0.333

(P>0.05)

FTL8 CGGCGAACAGCCTGGTGTCTGGGG (108a)

CGGCGAACAGCCTGGTGACTGGGG (108b)

108a/108a (9)

108a/108b (25)

108b/108b (4)

5.105

(P>0.05)

FTL12 TGGCAAGGAGTTCCGTTCCCTCGG (1012a)

TGGCAAGGAGTTCCGTTACCTCGG (1012b)

1012a/1012a (9)

1012a/1012b (15)

1012b/1012b (10)

0.529

(P>0.05)

Os 2-12 FTL7 TCGGCGCGTCGGCGCTGCCTGAGG (127a)

TCGGCGCGTCGGCGCTGTCTGAGG (127b)

127a/127a (6)

127a/127b (19)

127b/127b (9)

1.000

(P>0.05)

FTL8 CGGCGAACAGCCTGGTGTCTGGGG (128a)

CGGCGAACAGCCTGGTGCCTGGGG (128b)

128a/128a (8)

128a/128b (15)

128b/128b (9)

0.187

(P>0.05)

FTL12* TGGCAAGGAGTTCCGTTACCTCGG (1212a)

TGGCAAGGAGTTCCGTT-CCTCGG (1212b =WT)

1212a/1212a (13)

1212a/1212b (11)

1212b /1212b (3)

8.333

(P<0.05)

Os 2-13 FTL7 TCGGCGCGTCGGCGCT-CTGAGG (137a)

137a/137a (8)

(homozygote)

FTL8 CGGCGAACAGCCTGGTGACTGGGG (138a)

138a/138a (7)

(homozygote)

*In Os2-12 T0 plant, the edited site was heterozygous, and two individuals of this T1 line with new mutations were

detected:

Plant-1: TGGCAAGGAGTTCCGTTACCTCGG (1212a)

TGGCAAGGAGTTCCGTTTCCTCGG (1212c)

Plant-2: TGGCAAGGAGTTCCGTTACCTCGG (1212a)

TGGCAAGGAGTTCCGT--CCTCGG (1212d)

32

Supplementary Table 7. Inheritance of the varied alleles of At1g16210 in Arabidopsis T2 lines

determined by sequencing.

Line T1 genotype (code) T1 genotype

(No. plants)

At 4-2 GGCGGAGGTCGCCAAATCTCGTAAGAACGCCGCGGAGGCTGAGC (42a

=WT)

GGCGGAGG------------------------------CTGAGC (42b)

42a/42a (8)

42a/42b (9)

42b/42b (0 )

New mutations

(18)

At 4-15 ATCTCGTAAGAACGCCG-CGGAGG (415a =WT)

ATCTCGTAAGAACGCCGTCGGAGG (415b)

415a/415a (20)

415a/415b (9)

415b/415b (1)

New mutations (9)



ATCTCGTAAGAA----G-CGGAGG (436c)

436a/436a (6)

436a/436b (4)

436b/436b (1)

New mutations

(10)



ATCTCGTAAGAACG------GAGG (451c)

451a/451a (6)

451a/451b (4)

451b/451b (1)

451a/451c (1)

451c/451c (1)

451b/451c (4)

New mutations

(14)

Supplementary Table 8. Primers used for expression analysis.

Primer name Primer sequences (5’-3’) Purpose

Actin1-RT GAATAAACCGAACATATGTGTC RT of OsActin1

Actin2-R GTGGATTCCAGCAGCTTCCAT RT of AtActin2

C22 AGGAGGGTGAGGTCCTGGTGGTGCTCGTC RT of Cas9p

gRNA-RT CGACTCGGTGCCACTTTTTCAAGTTG RT of sgRNA & qPCR of sgRNAs

together with target-specific primers

Actin1-F CACATTCCAGCAGATGTGGA RT-qPCR of OsActin1 (Control)

Actin1-R ACCACAGGTAGCAATAGGTA

Actin2-F GCTGAGAGATTCAGATGCCCA RT-qPCR of AtActin2 (Control)

Actin2-R GTGGATTCCAGCAGCTTCCAT

Cas9RT-F CGCTCAGATTGGAGATCAGT RT-qPCR of Cas9p

Cas9RT-R CCTGGTGGTGCTCGTCGTAG

FTL1-U6a-F GCCGTCGGCCGGCAGCCGGATGA RT-qPCR of sgRNA targeting OsFTL1

FTL4-U6b-F GTTGTCACGAGGTAGGGATCCTT RT-qPCR of sgRNA targeting OsFTL4

FTL5-U6c-F TCAGACGCGCGGCTTGCCGGCGA RT-qPCR of sgRNA targeting OsFTL5

FTL6-U3-F GGCATCTTCACTAGCCATGTCAA RT-qPCR of sgRNA targeting OsFTL6

FTL9-U6c-F TCAGTATTGGTGGCACCGACCTGA RT-qPCR of sgRNA targeting OsFTL9

33

FTL10-U6a-F GCCGGCTTGCTTACAACTGCAGA RT-qPCR of sgRNA targeting OsFTL10

FTL11-U6b-F GTTGTCATGTCGTCGGCGATATCG RT-qPCR of sgRNA targeting OsFTL11

FTL13-U3-F GGCACGGTGCTGATGAAGGGATCC RT-qPCR of sgRNA targeting OsFTL13

Ara-2-F ATTGCCCTTTGGACGAACCCTCCA RT-qPCR of sgRNA targeting At1g56650

Documents

A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex targeting in plants · 2015-08-01 · 1 Supplementary Information A robust CRISPR/Cas9 system for convenient,