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Multiplex Gene Editing in Rice with Simplified CRISPR-Cpf1 and CRISPR-Cas9 Systems

Mugui Wang et al.

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Figure S1. Configuration of TCTU-1 and TCTU-2 systems for editing rice LEA_1 and LEA_2 family genes with FnCpf1

DR, direct repeat.

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Figure S2. Different configurations of crRNA expression cassette for LbCpf1 to edit the OsPDS-2 target site and the resultant mutation rate

The data in the first configuration was derived from our previous study (Wang et al. 2017). DR, direct repeat; HH, hammerhead ribozyme; HDV, hepatitis delta virus ribozyme; Bi, bi-allele; He, heterozygote; Chi, Chimera; WT, wild type.

Target 4353 (Bi) in plantlet #4

5’-CTGGTGGACAGGGCCAAGGGGTTCGTGGCGGAGAAGATCGCGAAAAT (Reference)

5’-CTGGTGGACAGGGCCA----------------GAAGATCGCGAAAAT (-16)

5’-CTGGTGGACAGGGCCAAG--------GGCGGAGAAGATCGCGAAAAT (-8)

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Target 5071 (Bi) in plantlet #4

5’-CAGCGTGCGTCGGCCATGTCGAGCTTGATGGACAAGGCGAAAGGGTT (Reference)

5’-CAGCGTGCGTCGGCCA------GCTTGATGGACAAGGCGAAAGGGTT (-6)

5’-CAGCGTGCGTCGGCCA-------CTTGATGGACAAGGCGAAAGGGTT (-7)

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Target 1258 (Bi) in plantlet #4

5’-GAATCAGGGCAAGAAACAATGGCGCAGCTGGTGGACAAGGCGAAGGG (Reference)

5’-GAATCAGGGCAAGAAACA-------AGCTGGTGGACAAGGCGAAGGG (-7)

5’-GAATCAGGGCAAGAAACA---------CTGGTGGACAAGGCGAAGGG (-9)

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Target 6262 (He) in plantlet #10

5’-CGTCTTCGTCGGAGAACCCCACGGTGACGGAGCGTGGCGGCGGGAAG (Reference)

5’-CGTCTTCGTCGGAGAACCCCACGGTGACGGAGCGTGGCGGCGGGAAG (WT)

5’-CGTCTTCGTCGGAGAACCCCA----------GCGTGGCGGCGGGAAG (-10)

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Figure S3. Representative mutations in targets 4353, 5071, 1258 and 6262 derived from the TCTU system of FnCpf1

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. PAM-guide sequence is marked in grey and the PAM motif (TTN or TTTV) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Bi: bi-allele, He: heterozygous, WT: wild type.

Target 4998 (He) in plantlet #16

5’-AGACGGCGAAGGTGAAGGTGCAGGACATGGTGAGCGGTGCCAAGGAG (Reference)

5’-AGACGGCGAAGGTGAAGGTGCAGGACATGGTGAGCGGTGCCAAGGAG (WT)

5’-AGACGGCGAAGGTGAAGGT-----ACATGGTGAGCGGTGCCAAGGAG (-5)

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Target 2387 (Bi) in plantlet #24

5’-TGAGTTTAGGTAGCCAGCATGCAGGCCGGGAAGAACGCGATGCAGTC (Reference)

5’-TGAGTTTAGGTAGCCAGCATGCA--------AGAACGCGATGCAGTC (-8)

5’-TGAGTTTAGGTAGCCAGCATGCA-----------------TGCAGTC (-17)

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Target 2191 (Bi) in plantlet #3

5’-TGGCTTTCATGGTCGCCATCCTCTCCCAAGTCCCAACTAGTCTTTAA (Reference)

5’-TGGCTTTCATGGTC----------------------------TTTAA (-28)

5’-TGGCTTTCATGGTCGCCATC----------------CTAGTCTTTAA (-16)

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Figure S4. Representative mutations in targets 4998, 2387 and 2191 derived from the TCTU system of FnCpf1

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. PAM-guide sequence is marked in grey and the PAM motif (TTN or TTTV) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Bi: bi-allele, He: heterozygous, WT: wild type.

Target 4353 (Bi) in plantlet #5

5’-CTGGTGGACAGGGCCAAGGGGTTCGTGGCGGAGAAGATCGCGAAAAT (Reference)

5’-CTGGTGGACAGGGCCAAG---------GCGGAGAAGATCGCGAAAAT (-9)

5’-CTGGTGGACAGGGCCAAG----------CGGAGAAGATCGCGAAAAT (-10)

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Target 5071 (He) in plantlet #5

5’-CAGCGTGCGTCGGCCATGTCGAGCTTGATGGACAAGGCGAAAGGGTT (Reference)

5’-CAGCGTGCGTCGGCCATGTCGAGCTTGATGGACAAGGCGAAAGGGTT (WT)

5’-CAGCGTGCGTCGGCC-----------GATGGACAAGGCGAAAGGGTT (-11)

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Target 1258 (He) in plantlet #13

5’-GAATCAGGGCAAGAAACAATGGCGCAGCTGGTGGACAAGGCGAAGGG (Reference)

5’-GAATCAGGGCAAGAAACAATGGCGCAGCTGGTGGACAAGGCGAAGGG (WT)

5’-GAATCAGGGCAAGAAACA------CAGCTGGTGGACAAGGCGAAGGG (-6)

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Target 6262 (He) in plantlet #23

5’-CGTCTTCGTCGGAGAACCCCACGGTGACGGAGCGTGGCGGCGGGAAG (Reference)

5’-CGTCTTCGTCGGAGAACCCCACGGTGACGGAGCGTGGCGGCGGGAAG (WT)

5’-CGTCTTCGTCGGAGAACCCCA--------GAGCGTGGCGGCGGGAAG (-8)

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Figure S5. Representative mutations in targets 4353, 5071, 1258 and 6262 derived from the SSTU system of FnCpf1

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. PAM-guide sequence is marked in grey and the PAM motif (TTN or TTTV) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Bi: bi-allele, He: heterozygous, WT: wild type.

Target 4998 (He) in plantlet #5

5’-AGACGGCGAAGGTGAAGGTGCAGGACATGGTGAGCGGTGCCAAGGAG (Reference)

5’-AGACGGCGAAGGTGAAGGTGCAGGACATGGTGAGCGGTGCCAAGGAG (WT)

5’-AGACGGCGAAGGTGAAG----------TGGTGAGCGGTGCCAAGGAG (-10)

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Target 2387 (He) in plantlet #20

5’-TGAGTTTAGGTAGCCAGCATGCAGGCCGGGAAGAACGCGATGCAGTC (Reference)

5’-TGAGTTTAGGTAGCCAGCATGCAGGCCGGGAAGAACGCGATGCAGTC (WT)

5’-TGAGTTTAGGTAGCCAGCATGCAGG-----AAGAACGCGATGCAGTC (-5)

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Target 2191 (Bi) in plantlet #22

5’-TGGCTTTCATGGTCGCCATCCTCTCCCAAGTCCCAACTAGTCTTTAA (Reference)

5’-TGGCTTTCATGGTCGCCATCCT-------GTCCCAACTAGTCTTTAA (-7)

5’-TGGCTTTCATGGTCGCCATCCT--------------CTAGTCTTTAA (-14)

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Figure S6. Representative mutations in targets 4998, 2387 and 2191 derived from the SSTU system of FnCpf1

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. PAM-guide sequence is marked in grey and the PAM motif (TTN or TTTV) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Bi: bi-allele, He: heterozygous, WT: wild type.

Target 5070 (He) in plantlet #7

5’-TCAGTTTGCACTGGCATCTGTAGCTTCAGTAGAGATTGAGGAAGAAT (Reference)

5’-TCAGTTTGCACTGGCATCTGTAGCTTCAGTAGAGATTGAGGAAGAAT (WT)

5’-TCAGTTTGCACTGGCATC--------------AGATTGAGGAAGAAT (-14)

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Target 4487 (He) in plantlet #18

5’-AGCTTGTCACCGGCATGGAGAAGGTCTCCGTGGAAGAGCCAAAGAAG (Reference)

5’-AGCTTGTCACCGGCATGGAGAAGGTCTCCGTGGAAGAGCCAAAGAAG (WT)

5’-AGCTTGTCACCGGCATGGAGA---TCTCCGTGGAAGAGCCAAAGAAG (-3)

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Target 4528-2 (Bi) in plantlet #19

5’-TCAATTTCTTGCACAACAACTACCTGCATGCACGCACGCACGGCGCC (Reference)

5’-TCAATTTCTTGCACAACAACTAC----------GCACGCACGGCGCC (-10)

5’-TCAATTTCTTGCACAACAACTAC-----------------CGGCGCC (-17)

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Target 2657 (He) in plantlet #24

5’-GTGGTTTAATGCCTGGGCACACTGCCGGGCATGGTACCACCGGCACC (Reference)

5’-GTGGTTTAATGCCTGGGCACACTGCCGGGCATGGTACCACCGGCACC (WT)

5’-GTGGTTTAATGCCTGGGCACACTGC------TGGTACCACCGGCACC (-6)

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Figure S7. Representative mutations in targets 5070, 4487, 4528-2 and 2657 derived from the TCTU system of LbCpf1

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. PAM-guide sequence is marked in grey and the PAM motif (TTTV) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Bi: bi-allele, He: heterozygous, WT: wild type.

Target 2675 (He) in plantlet #7

5’-GCCGCGCCGACGAGCACGGCAACCCTGCAGTGACCACCGGAAACGCA (Reference)

5’-GCCGCGCCGACGAGCACGGCAACCCTGCAGTGACCACCGGAAACGCA (WT)

5’-GCCGCGCCGACGAGCAC-----------AGTGACCACCGGAAACGCA (-11)

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Target 2676 (Bi) in plantlet #18

5’-CGTGTTTGAGAAGATGGAGAACTACCAGGGGCAGCACGGCTACGGCG (Reference)

5’-CGTGTTTGAGAAGATG------------------CACGGCTACGGCG (-18)

5’-CGTGTTTGAGAAGATGGA------------GCAGCACGGCTACGGCG (-12)

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Target 2678 (Ho) in plantlet #1

5’-TTAATTTGCTTGAGAGGATGGACAACTACCAGGGGCAGCACGGCTAC (Reference)

5’-TTAATTTGCTTGAGAGG-----------------------CGGCTAC (-23)

5’-TTAATTTGCTTGAGAGG-----------------------CGGCTAC (-23)

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Target 2679 (Bi) in plantlet #12

5’-CAGCTTTAGCTTAAGCAGGATGGAGCACCAGGGGCAGCACGGCCACG (Reference)

5’-CAGCTTTAGCTTAAGCAGGATG-------AGGGGCAGCACGGCCACG (-7)

5’-CAGCTTTAGCTTAAGCAGGATG--------GGGGCAGCACGGCCACG (-8)

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Figure S8. Representative mutations in targets 2675, 2676, 2678 and 2679 derived from the TCTU system of LbCpf1

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. PAM-guide sequence is marked in grey and the PAM motif (TTTV) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Ho: homozygous, Bi: bi-allele, He: heterozygous, WT: wild type.

Target 5070 (He) in plantlet #7

5’-TCAGTTTGCACTGGCATCTGTAGCTTCAGTAGAGATTGAGGAAGAAT (Reference)

5’-TCAGTTTGCACTGGCATCTGTAGCTTCAGTAGAGATTGAGGAAGAAT (WT)

5’-TCAGTTTGCACTGGCATCTGTAGC---AGTAGAGATTGAGGAAGAAT (-3)

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Target 4487 (Bi) in plantlet #5

5’-AGCTTGTCACCGGCATGGAGAAGGTCTCCGTGGAAGAGCCAAAGAAG (Reference)

5’-AGCTTGTCACCGGCATG------GTCTCCGTGGAAGAGCCAAAGAAG (-6)

5’-AGCTTGTCACCGGCATGCA--------CCGTGGAAGAGCCAAAGAAG (-8)

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Target 4528-2 (Bi) in plantlet #20

5’-TCAATTTCTTGCACAACAACTACCTGCATGCACGCACGCACGGCGCC (Reference)

5’-TCAATTTCTTGCACAACAACTAC----ATGCACGCACGCACGGCGCC (-4)

5’-TCAATTTCTTGCACAACAACTAC----------GCACGCACGGCGCC (-10)

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Target 2657 (He) in plantlet #5

5’-GTGGTTTAATGCCTGGGCACACTGCCGGGCATGGTACCACCGGCACC (Reference)

5’-GTGGTTTAATGCCTGGGCACACTGCCGGGCATGGTACCACCGGCACC (WT)

5’-GTGGTTTAATGCCTGGGCA-A-----------------------ACC (-24)

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Figure S9. Representative mutations in targets 5070, 4487, 4528-2 and 2657 derived from the SSTU system of LbCpf1

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. PAM-guide sequence is marked in grey and the PAM motif (TTTV) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Bi: bi-allele, He: heterozygous, WT: wild type.

Target 2675 (He) in plantlet #11

5’-GCCGCGCCGACGAGCACGGCAACCCTGCAGTGACCACCGGAAACGCA (Reference)

5’-GCCGCGCCGACGAGCACGGCAACCCTGCAGTGACCACCGGAAACGCA (WT)

5’-GCCGCGCCGACGAGCAC-------CTGCAGTGACCACCGGAAACGCA (-7)

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Target 2676 (Bi) in plantlet #11

5’-CGTGTTTGAGAAGATGGAGAACTACCAGGGGCAGCACGGCTACGGCG (Reference)

5’-CGTGTTTGAGAAGATGGAGAA------------GCACGGCTACGGCG (-12)

5’-CGTGTTTGAGAAGATGGAGAACT-------GCAGCACGGCTACGGCG (-7)

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Target 2678 (Bi) in plantlet #14

5’-TTAATTTGCTTGAGAGGATGGACAACTACCAGGGGCAGCACGGCTAC (Reference)

5’-TTAATTTGCTTGAGAGGATGGAC---TACCAGGGGCAGCACGGCTAC (-3)

5’-TTAATTTGCTTGAGAGGATGGACAA----CAGGGGCAGCACGGCTAC (-4)

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Target 2679 (Bi) in plantlet #6

5’-CAGCTTTAGCTTAAGCAGGATGGAGCACCAGGGGCAGCACGGCCACG (Reference)

5’-CAGCTTTAGCTTAAGCA-------------------GCACGGCCACG (-19)

5’-CAGCTTTAGCTTAAGCA----------------------CGGCCACG (-22)

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Figure S10. Representative mutations in targets 2675, 2676, 2678 and 2679 derived from the SSTU system of LbCpf1

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. PAM-guide sequence is marked in grey and the PAM motif (TTTV) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Bi: bi-allele, He: heterozygous, WT: wild type.

Target 230 (Bi) in plantlet #6

5’-TGAAGTACTCCACTTCTGTAACCATGGATAAGGCCTACATTGCCGTC (Reference)

5’-TGAAGTACTCCACTTCTGTA-CCATGGATAAGGCCTACATTGCCGTC (-1)

5’-TGAAGTACTCCACTTCTGTAA--ATGGATAAGGCCTACATTGCCGTC (-2)

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Target 240 (Bi) in plantlet #11

5’-TCCAGCTGAAGCGGAGGCTGTT-CGAGGTCTCCCTCAGCGTGCTCATG (Reference)

5’-TCCAGCTGAAGCGGAGGCT-TT-CGAGGTCTCCCTCAGCGTGCTCATG (-1)

5’-TCCAGCTGAAGCGGAGGCTGTTTCGAGGTCTCCCTCAGCGTGCTCATG (+1)

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Target 250 (Ho) in plantlet #18

5’-GGCAGCAACAATGGCGAAAACAAGGGGATGTTGCAGCTGCCTCCGAG (Reference)

5’-GGCAGCAACAATGGCG---ACAAGGGGATGTTGCAGCTGCCTCCGAG (-3)

5’-GGCAGCAACAATGGCG---ACAAGGGGATGTTGCAGCTGCCTCCGAG (-3)

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Figure S11. Representative mutations derived from the TCTU-3 system of Cas9

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. Guide-PAM sequence is marked in grey and the PAM motif (NGG) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ and ‘+’ represents the number of bases that have been deleted and inserted, respectively. Ho: homozygous, Bi: bi-allele.

Target 230 (Ho) in plantlet #1

5’-TGAAGTACTCCACTTCTGTAACCATGGATAAGGCCTACATTGCCGTC (Reference)

5’-TGAAGTACTCCACTTCTGTAA---TGGATAAGGCCTACATTGCCGTC (-3)

5’-TGAAGTACTCCACTTCTGTAA---TGGATAAGGCCTACATTGCCGTC (-3)

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Target 240 (Bi) in plantlet #17

5’-TCCAGCTGAAGCGGAGGCTGTTCGAGGTCTCCCTCAGCGTGCTCATG (Reference)

5’-TCCAGCTGAAGCGGAGGCT-TTCGAGGTCTCCCTCAGCGTGCTCATG (-1)

5’-TCCAGCTGAAGCGGAGGCT--TCGAGGTCTCCCTCAGCGTGCTCATG (-2)

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Target 250 (He) in plantlet #15

5’-GGCAGCAACAATGGCGAAAACAAGGGGATGTTGCAGCTGCCTCCGAG (Reference)

5’-GGCAGCAACAATGGCGAAAACAAGGGGATGTTGCAGCTGCCTCCGAG (WT)

5’-GGCAGCAACAATGGCGAAAA--AGGGGATGTTGCAGCTGCCTCCGAG (-2)

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Target 260 (Ho) in plantlet #3

5’-AGCGGCATGCCATTGCTCCTGCCGCGGCGGCGGAAGAGGAAGTGAAT (Reference)

5’-AGCGGCATGCCATTGCTCCT-CCGCGGCGGCGGAAGAGGAAGTGAAT (-1)

5’-AGCGGCATGCCATTGCTCCT-CCGCGGCGGCGGAAGAGGAAGTGAAT (-1)

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Figure S12. Representative mutations derived from the SSTU system of Cas9

The genotypes, detail on mutations and original sequencing chromatograms for each target site are listed. Guide-PAM sequence is marked in grey and the PAM motif (NGG) is marked in bold. Each dashed line represents a deleted nucleotide and the number after ‘-’ represents the number of bases that have been deleted. Ho: homozygous, Bi: bi-allele, He: heterozygous, WT: wild type.

Table S1. Summary of the multiplex gene editing in rice LEA_1 and LEA_2 family genes by TCTU-1 and TCTU-2 systems with FnCpf1

|Target gene and PAM-guide sequence (5’-3’) |Constructions |Mutation rate |Genotype of the plantlets |

|LOC_Os01g43530 |TCTU-1 |0/24 (0%) |24WT |

|TTTTCGCGATCTTCTCCGCCACGAAC |TCTU-2 |0/24 (0%) |24WT |

|LOC_Os05g50710 |TCTU-1 |0/24 (0%) |24WT |

|TTTCGCCTTGTCCATCAAGCTCGACAT |TCTU-2 |0/24 (0%) |24WT |

|LOC_Os01g12580 |TCTU-1 |1/24 (4.2%) |1He+23WT |

|TTCGCCTTGTCCACCAGCTGCGCCAT |TCTU-2 |10/24 (41.2%) |1Bi+2He+7Chi+14WT |

|LOC_Os03g62620 |TCTU-1 |0/24 (0%) |24WT |

|TTCGTCGGAGAACCCCACGGTGACGGA |TCTU-2 |4/24 (16.7%) |2He+2Chi+20WT |

|LOC_Os04g49980 |TCTU-1 |2/24 (8.3%) |1He+1Chi+22WT |

|TTGGCACCGCTCACCATGTCCTGCACCT |TCTU-2 |12/24 (50.0%) |3Bi+4He+5Chi+12WT |

|LOC_Os06g02040 |TCTU-1 |0/24 (0%) |24WT |

|TTCACCGCGCTCACCTTGTCCTTCACCT |TCTU-2 |2/24 (8.3%) |2Chi+22WT |

|LOC_Os08g23870 |TCTU-1 |0/24 (0%) |24WT |

|TTGGTCGACTGCATCGCGTTCTTCCCG |TCTU-2 |1/24 (4.2%) |1He+23WT |

|LOC_Os06g21910 |TCTU-1 |0/24 (0%) |24WT |

|TTGTGGACGCCGGCCTTGGCGGAGGAT |TCTU-2 |0/24 (0%) |24WT |

Note: He, heterozygote; Chi, Chimera; WT, wild-type. The TTN PAM is marked in bold.

Table S2. Summary of the multiplex gene editing in rice LEA_1 and LEA_2 family genes by TCTU and SSTU systems with FnCpf1

|Target gene and PAM-guide sequence (5’-3’) |Constructions |Mutation rate |Genotype of the plantlets |

|LOC_Os01g43530 |TCTU |16/24 (66.7%) |1Ho+15Bi+8WT |

|TTTCGCGATCTTCTCCGCCACGAACCC |SSTU |17/24 (70.8%) |10Bi+2He+5Chi+7WT |

|LOC_Os05g50710 |TCTU |13/24 (54.2%) |1Ho+8Bi+2He+2Chi+11WT |

|TTTCGCCTTGTCCATCAAGCTCGACATG |SSTU |12/24 (50.0%) |7He+5Chi+12WT |

|LOC_Os01g12580 |TCTU |13/24 (54.2%) |4Bi+6He+3Chi+11WT |

|TTCGCCTTGTCCACCAGCTGCGCCAT |SSTU |8/24 (33.3%) |5He+3Chi+16WT |

|LOC_Os03g62620 |TCTU |10/24 (41.7%) |1Bi+6He+3Chi+14WT |

|TTCGTCGGAGAACCCCACGGTGACGGA |SSTU |7/24 (29.2%) |3He+4Chi+17WT |

|LOC_Os04g49980 |TCTU |7/24 (29.2%) |1Bi+4He+2Chi+17WT |

|TTGGCACCGCTCACCATGTCCTGCACCT |SSTU |3/24 (12.5%) |2He+1Chi+21WT |

|LOC_Os06g02040 |TCTU |0/24 (0%) |24WT |

|TTCACCGCGCTCACCTTGTCCTTCA |SSTU |0/24 (0%) |24WT |

|LOC_Os08g23870 |TCTU |17/24 (70.8%) |1Ho+12Bi+2He+2Chi+7WT |

|TTTAGGTAGCCAGCATGCAGGCCGGGAA |SSTU |16/24 (66.7%) |1Ho+7Bi+2He+6Chi+8WT |

|LOC_Os06g21910 |TCTU |16/24 (66.7%) |10Bi+5He+1Chi+8WT |

|TTTCATGGTCGCCATCCTCTCCCAAGTC |SSTU |12/24 (50.0%) |5Bi+3He+4Chi+12WT |

Note: Ho, homozygote; Bi, bi-allele; He, heterozygote; Chi, Chimera; WT, wild-type. The TTN or TTTV PAM is marked in bold.

Table S3. Summary of the multiplex gene editing in rice LEA_Dehydrin family genes by TCTU and SSTU systems with LbCpf1

|Target gene and PAM-guide sequence (5’-3’) |Constructions |Mutation rate |Genotype of the plantlets |

|LOC_Os01g50700 |TCTU |8/24 (33.3%) |1Bi+6He+1Chi+16WT |

|TTTGCACTGGCATCTGTAGCTTCAGTAG |SSTU |9/24 (37.5%) |2Bi+5He+2Chi+15WT |

|LOC_Os02g44870 |TCTU |4/24 (16.7%) |3He+1Chi+20WT |

|TTTGGCTCTTCCACGGAGACCTTCTCCA |SSTU |4/24 (16.7%) |1Bi+3Chi+20WT |

|LOC_Os03g45280(1) |TCTU |11/24 (45.8%) |2Bi+3He+6Chi+13WT |

|TTTCTCTTGAGACCGTAGAGAGAGAGA |SSTU |15/24 (62.5%) |4Ho+4Bi+5He+2Chi+9WT |

|LOC_Os03g45280(2) |TCTU |13/24 (54.2%) |5Bi+3He+5Chi+11WT |

|TTTCTTGCACAACAACTACCTGCATGC |SSTU |17/24 (70.8%) |2Ho+7Bi+5He+3Chi+7WT |

|LOC_Os11g26570 |TCTU |1/24 (4.2%) |1He+23WT |

|TTTAATGCCTGGGCACACTGCCGGGCAT |SSTU |2/24 (8.3%) |1He+1Chi+22WT |

|LOC_Os11g26750 |TCTU |5/24 (20.8%) |4He+1Chi+19WT |

|TTTCCGGTGGTCACTGCAGGGTTGCCGT |SSTU |1/24 (4.2%) |1He+23WT |

|LOC_Os11g26760 |TCTU |11/24 (45.8%) |5Bi+4He+2Chi+13WT |

|TTTGAGAAGATGGAGAACTACCAGGGG |SSTU |9/24 (37.5%) |4Bi+3He+2Chi+15WT |

|LOC_Os11g26780 |TCTU |13/24 (54.2%) |4Ho+6Bi+1He+2Chi+11WT |

|TTTGCTTGAGAGGATGGACAACTACCA |SSTU |14/24 (58.3%) |2Ho+3Bi+7He+2Chi+10WT |

|LOC_Os11g26790 |TCTU |13/24 (54.2%) |2Ho+10Bi+1He+11WT |

|TTTAGCTTAAGCAGGATGGAGCACCAG |SSTU |14/24 (58.3%) |1Ho+9Bi+1He+3Chi+10WT |

Note: Ho, homozygote; Bi, bi-allele; He, heterozygote; Chi, Chimera; WT, wild-type. The TTTV PAM is marked in bold.

Table S4. Mutants derived from the TCTU system of FnCpf1

| Target loci |4353 |5071 |1258 |

|No. plantlets | | | |

|LOC_Os03g55230 |TCTU-3 |19/24 (79.2%) |5Ho+11Bi+1He+2Chi+5WT |

|GTACTCCACTTCTGTAACCATGG |SSTU |19/22 (86.4%) |4Ho+11Bi+1He+3Chi+3WT |

|LOC_Os03g55240 |TCTU-3 |17/24 (70.8%) |6Ho+7Bi+1He+3Chi+7WT |

|GCTGAAGCGGAGGCTGTTCGAGG |SSTU |18/22 (81.8%) |1Ho+3Bi+10He+4Chi+4WT |

|LOC_Os03g55250 |TCTU-3 |17/24 (70.8%) |2Ho+7Bi+1He+7Chi+7WT |

|A(G)CAACAATGGCGAAAACAAGGGG |SSTU |11/22 (50.0%) |1Bi+5He+5Chi+11WT |

|LOC_Os03g55260 |TCTU |19/22 (86.4%) |4Ho+12Bi+2He+1Chi+3WT |

|GCATGCCATTGCTCCTGCCGCGG | | | |

Note: guide 250 was begun with A in TCTU-3 system and begun with G in SSTU system. Ho, homozygote; Bi, bi-allele; He, heterozygote; Chi, Chimera; WT, wild-type. The NGG PAM for Cas9 is marked in bold.

Table S9. Mutants derived from the TCTU-3 system of Cas9

| Target loci |230 |250 |240 |

|No. plantlets | | | |

|#1 |Bi |WT |WT |

|#2 |Bi |Chi |Bi |

|#3 |Bi |Chi |Ho |

|#4 |Bi |Bi |Bi |

|#5 |Bi |Bi |Bi |

|#6 |Bi |He |Ho |

|#7 |Bi |Bi |Bi |

|#9 |Bi |Ho |Ho |

|#10 |Chi |Chi |Chi |

|#11 |Bi |Chi |Bi |

|#13 |Chi |WT |WT |

|#14 |Ho |Bi |Ho |

|#15 |Ho |Chi |Ho |

|#16 |Bi |Chi |Chi |

|#17 |Ho |Bi |Bi |

|#18 |Ho |Ho |He |

|#19 |Bi |Bi |Bi |

|#22 |He |Bi |Chi |

|#24 |Ho |Chi |Ho |

Note: Ho, homozygote; Bi, bi-allele; He, heterozygote; Chi, Chimera; WT, wild-type.

Table S10. Mutants derived from the SSTU system of Cas9

| Target loci |260 |230 |250 |240 |

|No. plantlets | | | | |

|#1 |Ho |Ho |Chi |Chi |

|#2 |Ho |Ho |Bi |He |

|#3 |Ho |Bi |He |Ho |

|#4 |Bi |Chi |WT |He |

|#5 |Chi |Bi |He |He |

|#6 |Bi |Bi |WT |He |

|#7 |Bi |Bi |WT |He |

|#9 |Bi |He |Chi |Chi |

|#10 |Bi |Bi |Chi |He |

|#11 |Bi |Bi |WT |He |

|#12 |He |WT |WT |WT |

|#13 |Bi |Bi |WT |WT |

|#14 |WT |Ho |Chi |He |

|#15 |Bi |Bi |He |He |

|#16 |Bi |Ho |WT |Chi |

|#17 |He |Bi |WT |Bi |

|#18 |Bi |Chi |He |Chi |

|#20 |Bi |Bi |Chi |Bi |

|#21 |Ho |Chi |WT |Bi |

|#22 |Bi |Bi |He |He |

Note: Ho, homozygote; Bi, bi-allele; He, heterozygote; Chi, Chimera; WT, wild-type.

Table S11. PCR primers used to amplify the target sites in rice plantlets

|Target |Forward primer (5' to 3') |Reverse primer (5' to 3') |PCR product |

| | | |(bp) |

|LOC_Os03g55230 |ACCCTACATGTTTAAGGACCG |TCGAACAGCCTCCGCTTCAG |986 |

|LOC_Os03g55240 |CAGCTGCTGGTCTCGTTCAA |TGCTGAATGCTGATAGTGCC |806 |

|LOC_Os03g55250 |GTAGCTGCTATGCGTAATACAAC |GCTATGGCTTCCATGAGGAC |645 |

|LOC_Os03g55260 |GTTTTATAGTTATTACGCACCAGG |GGCTTCCATGAGGACGCTGA |864 |

|LOC_Os01g43530 |ATGGTAACGCAGGCAGAACA |ATAATGCACGCACTTGTCGC |412 |

|LOC_Os05g50710 |TCAAATTCCCCTTGCAGCCA |ATCGAGCGTGAGATGGATGG |600 |

|LOC_Os01g12580 |ACACTCGTCGAGCGGTTAG |AGACACCGCGCATACGATTC |783 |

|LOC_Os03g62620 |GCTCTCGCTTCGTTTTGGAG |GATAGGGACCGGGTTAGGGT |497 |

|LOC_Os04g49980 |GTCGCAAGTCGAAGCACAAA |AGATCCGTAGATGCGTACTTCA |733 |

|LOC_Os06g02040 |CCATCATACGCACTGCAAGC |TGCACACTGGCTGGCTTATT |499 |

|LOC_Os08g23870 |CACAACACACATCGCCCATC |ATGAGAATGAGATCACTGGTACTG |672 |

|LOC_Os06g21910 |CCGATCGAGCAGATCAGACA |GGTGGTAAAGGTGTGCTTGC |383 |

|LOC_Os01g50700 |AGGAACTGGGTGAGGCCTAT |AAGATGGAGGGCAATACCCG |740 |

|LOC_Os02g44870 |CAAAAGCGACTCGTCACAGC |CACCCTCGATCTTCTCCACG |742 |

|LOC_Os03g45280 |GAACATCACCACAGCTAGATCA |ATCATGTCAGTCACACGCCAA |519 |

|LOC_Os11g26570 |TGATCAACTCATCGCCTCTCG |GTATGCCATGGTGTCGGGTG |647 |

|LOC_Os11g26750 |TCCATTCAGTGCCAATCAGGT |AAAAGTGGCTACAATCCGAACA |362 |

|LOC_Os11g26760 |TTCCGTACCCTTCCTGACCT |ACGCACACGAGTACAACTCA |492 |

|LOC_Os11g26780 |ATCGATCGACGGCTTTGACA |ACAGTGAACGTACCGAGCTG |382 |

|LOC_Os11g26790 |GCACACGTCTCCCTGTCTC |TCAACGAATTTGTACGCAAGGT |521 |

Table S12. Primers used to sequence the target PCR product

|Target |Primer (5' to 3') |

|LOC_Os03g55230 |ACCCTACATGTTTAAGGACCG |

|LOC_Os03g55240 |CAGCTGCTGGTCTCGTTCAA |

|LOC_Os03g55250 |GTAGCTGCTATGCGTAATACAAC |

|LOC_Os03g55260 |GGCTTCCATGAGGACGCTGA |

|LOC_Os01g43530 |TGTACCGATCCGTCACGAC |

|LOC_Os05g50710 |TCAAATTCCCCTTGCAGCCA |

|LOC_Os01g12580 |ACACTCGTCGAGCGGTTAG |

|LOC_Os03g62620 |GCTCTCGCTTCGTTTTGGAG |

|LOC_Os04g49980 |GTCGCAAGTCGAAGCACAAA |

|LOC_Os06g02040 |CCATCATACGCACTGCAAGC |

|LOC_Os08g23870 |CGTTGCTTGCCGCTCTATAA |

|LOC_Os06g21910 |GCAATTTGGCCGGTCATGAT |

|LOC_Os01g50700 |AGGAACTGGGTGAGGCCTAT |

|LOC_Os02g44870 |CAAAAGCGACTCGTCACAGC |

|LOC_Os03g45280(1) |GAACATCACCACAGCTAGATCA |

|LOC_Os03g45280(2) |ATCATGTCAGTCACACGCCAA |

|LOC_Os11g26570 |TGATCAACTCATCGCCTCTCG |

|LOC_Os11g26750 |TCCATTCAGTGCCAATCAGGT |

|LOC_Os11g26760 |TTCCGTACCCTTCCTGACCT |

|LOC_Os11g26780 |ATCGATCGACGGCTTTGACA |

|LOC_Os11g26790 |GCACACGTCTCCCTGTCTC |

Materials and Methods

Vector construction

The CRISPR/FnCpf1 and CRISPR/LbCpf1 vectors with the Cpf1 expression cassette and OsU6 driven sgRAN expression cassette were developed in our previous study (Wang et al. 2017a). Pol II driven cassettes for sgRAN expression were constructed as follows: The Actin-NOS cassette was amplified from a common used over expression plasmid in rice and the Ubi-NOS cassette was amplified from the CRISPR/Cpf1 itself. The CmYLCV promoter was synthesized according to the sequence provided in the study of Čermák et al. (2017). The above three cassettes were then cloned into the Hind III restriction site of our previous CRISPR/FnCpf1 or CRISPR/LbCpf1 system to replace the OsU6-sgRAN expression cassette, respectively. The sequence of hammerhead (HH) and hepatitis delta virus (HDV) ribozyme, or tRANGly were designed according to the study of Gao and Zhao (2014) or Čermák et al. (2017). For single gene editing in the OsPDS-2 target site, the DR-guide sequence with or without another DR or HH-HDV were synthesized and cloned into the Bsa I restriction site in OsU6-sgRAN cassette, or the multi-cloning sites in Pol II driven sgRAN expression cassettes. For multiplex gene editing, the crRNA array that includes several mature DRs and 22-25 bp guide sequences, with or without HH-HDV and tRANGly elements were synthesized by Sangon Biotech (Shanghai, China) and cloned into the Bsa I restriction site in OsU6-sgRAN cassette (TCTU-1 and TCTU-2), or the multi-cloning sites in Pol II driven sgRAN expression cassettes (TCTU). To construct SSTU systems for both FnCpf1 and LbCpf1, the synthesized arrays were directly cloned into the 3’ UTR of Cpf1 expression cassette via BamHI restriction site. Target genes in rice late embryogenesis abundant (LEA) family were classified according to Wang et al (2007). All guides were designed using the online tool Cas-OFFinder (Bae et al. 2014) to avoid potential off-target sites.

The CRISPR/Cas9 vector was also derived from our previous study (Wang et al. 2017b). To design the targets for loci 230, 240, 250 and 260 in rice CYP81A family, the 23-bp targeting sequences (guide and PAM) were selected within the target genes and their targeting specificity were confirmed at CRISPR-P (). To construct the TCTU-3 system, three sgRNA expression plasmids were developed, each contain one of the following three pol III promoters in the pUC19 backbone: OsU6, OsU31 and OsU62. These plasmids were named as pUC-U6, pUC-U31 and pUC-U62, respectively. Three pair of oligos for guide230, 250 and 240 were synthesized and annealed to form a duplex short fragment with adaptors and ligated into pUC-U6, pUC-U31 and pUC-U62, respectively, as described in our previous study (Feng et al. 2013). At last, the above three sgRNA expression cassettes were PCR amplified and recombinated into the CRISPR/Cas9 vector using EasyGeno Assembly Cloning kit (TIANGEN, China) according to manufacturer’s instruction. For SSTU system construction, a pair of oligos for guide260 were designed and cloned into the OsU6-sgRNA expression cassette in CRISPR-Cas9 vector as described in our previous study (Feng et al. 2013). The resulted construct was named pCas9-260. The sgRNAs array for targeting loci 230, 250 and 240 was directly synthesized by Sangon Biotech (Shanghai, China) and cloned into the 3’ UTR of Cas9 expression cassette in pCas9-260 via BamHI restriction site. Detail architecture and sequence of the constructs are listed in Supplemental Sequences.

Rice transformation procedures

The constructs (all contain hygromycin-resistant gene) were introduced into Agrobacterium tumefaciens strain EHA105, and then transformed into embryogenic calli induced from rice Nipponbare (Oryza sativa L. ssp. japonica) mature seeds. Rice transformation, tissue culture and plantlets growth procedure were mainly performed as described previously (Nishimura et al. 2006; Wang et al. 2015).

Genotyping and sequence analysis of targeted mutations

After the hygromycin-resistant transgenic plantlets were generated, genomic DNA was isolated from 2-3 fresh leaves of each transgenic plantlets. The target loci were amplified by PCR and then the resulting products were analyzed by Sanger sequencing. The sequencing chromatograms were analyzed by DSDecodeM (Liu et al. 2015; Ma et al. 2015). Some samples with complicate chromatograms were further confirmed by TA cloning sequencing, where 10-20 positive colonies for each sample were sequenced. Mutation ratio was estimated by scoring the number of plantlets with mutation around the target site relative to the total number of identified plantlets. Primers used for the PCR and sequencing are listed in Tables S11-12.

Supplemental Sequences

crRNA array expression cassettes for LEA_2 and LEA_1 in FnCpf1 TCTU-1 system.

>OsU6-DR-g4353-DR-g5071-DR-g1258-DR-g6262-DR-g4998-DR-g0204-DR-g2387-DR-g2191-poly T.

GGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGTAATTTCTACTGTTGTAGATTCGCGATCTTCTCCGCCACGAACTAATTTCTACTGTTGTAGATCGCCTTGTCCATCAAGCTCGACATTAATTTCTACTGTTGTAGATGCCTTGTCCACCAGCTGCGCCATTAATTTCTACTGTTGTAGATGTCGGAGAACCCCACGGTGACGGATAATTTCTACTGTTGTAGATGCACCGCTCACCATGTCCTGCACCTTAATTTCTACTGTTGTAGATACCGCGCTCACCTTGTCCTTCACCTTAATTTCTACTGTTGTAGATGTCGACTGCATCGCGTTCTTCCCGTAATTTCTACTGTTGTAGATTGGACGCCGGCCTTGGCGGAGGATTAATTTCTACTGTTGTAGATTTTTTTT

Double crRNA array expression cassettes for LEA_2 and LEA_1 in FnCpf1 TCTU-2 system.

>OsU6-DR-g4353-DR-g5071-DR-g1258-DR-g6262-poly T-OsU6-DR-g4998-DR

-g0204-DR-g2387-DR-g2191-poly T.

GGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGTAATTTCTACTGTTGTAGATTCGCGATCTTCTCCGCCACGAACTAATTTCTACTGTTGTAGATCGCCTTGTCCATCAAGCTCGACATTAATTTCTACTGTTGTAGATGCCTTGTCCACCAGCTGCGCCATTAATTTCTACTGTTGTAGATGTCGGAGAACCCCACGGTGACGGATTTTTTTGGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGTAATTTCTACTGTTGTAGATGCACCGCTCACCATGTCCTGCACCTTAATTTCTACTGTTGTAGATACCGCGCTCACCTTGTCCTTCACCTTAATTTCTACTGTTGTAGATGTCGACTGCATCGCGTTCTTCCCGTAATTTCTACTGTTGTAGATTGGACGCCGGCCTTGGCGGAGGATTTTTTTT

crRNA array for LEA_2 and LEA_1 in FnCpf1 TCTU system

>HH-DR-g4353-DR-g5071-DR-g1258-DR-g6262-DR-g4998-DR-g0204-DR-g2387-DR-g2191-HDV.

TGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCTGCAGGGTACCAAATTACTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCTAATTTCTACTGTTGTAGATGCGATCTTCTCCGCCACGAACCCTAATTTCTACTGTTGTAGATGCCTTGTCCATCAAGCTCGACATGTAATTTCTACTGTTGTAGATGCCTTGTCCACCAGCTGCGCCATTAATTTCTACTGTTGTAGATGTCGGAGAACCCCACGGTGACGGATAATTTCTACTGTTGTAGATGCACCGCTCACCATGTCCTGCACCTTAATTTCTACTGTTGTAGATACCGCGCTCACCTTGTCCTTCATAATTTCTACTGTTGTAGATGGTAGCCAGCATGCAGGCCGGGAATAATTTCTACTGTTGTAGATATGGTCGCCATCCTCTCCCAAGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACGGTACCACTAGTGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTA

Note: The italic characters are Kpn I restriction sites for crRNA array cloning. Sequences in gray and orange are partial sequences of the Ubi promoter and NOS terminator, respectively.

crRNA array for LEA_2 and LEA_1 in FnCpf1-SSTU system

> DR-g4353-DR-g5071-DR-g1258-DR-g6262-DR-g4998-DR-g0204-DR-g2387-DR-

g2191-DR.

ACGAGGAATACTTCGAGTTTGTCCAGAATAGAAATAACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAAGGATCCGGTACCTAATTTCTACTGTTGTAGATGCGATCTTCTCCGCCACGAACCCTAATTTCTACTGTTGTAGATGCCTTGTCCATCAAGCTCGACATGTAATTTCTACTGTTGTAGATGCCTTGTCCACCAGCTGCGCCATTAATTTCTACTGTTGTAGATGTCGGAGAACCCCACGGTGACGGATAATTTCTACTGTTGTAGATGCACCGCTCACCATGTCCTGCACCTTAATTTCTACTGTTGTAGATACCGCGCTCACCTTGTCCTTCATAATTTCTACTGTTGTAGATGGTAGCCAGCATGCAGGCCGGGAATAATTTCTACTGTTGTAGATATGGTCGCCATCCTCTCCCAAGTCTAATTTCTACTGTTGTAGATGGTACCGGATCCTGATTGATCGATAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATAT

Note: NLS and stop coden TAA of the FnCpf1 expression cassette are marked in green and gray, respectively. Sequences before NLS belong to the CDS of FnCpf1. The Bam HI restriction sites for crRNA array cloning are marked in italic sequences. Characters in purple are partial sequences of the NOS terminator.

crRNA array expression cassettes for LEA_Dehydrin in LbCpf1 TCTU system.

>CmYLCV-tRANGly-DR-g5070-DR-g4487-DR-g4528(1)-DR-g4528(2)-DR-g2657-

DR-g2675-DR-g2676-DR-g2678-DR-g2679-DR-tRANGly-poly A

TGGCAGACATACTGTCCCACAAATGAAGATGGAATCTGTAAAAGAAAACGCGTGAAATAATGCGTCTGACAAAGGTTAGGTCGGCTGCCTTTAATCAATACCAAAGTGGTCCCTACCACGATGGAAAAACTGTGCAGTCGGTTTGGCTTTTTCTGACGAACAAATAAGATTCGTGGCCGACAGGTGGGGGTCCACCATGTGAAGGCATCTTCAGACTCCAATAATGGAGCAATGACGTAAGGGCTTACGAAATAAGTAAGGGTAGTTTGGGAAATGTCCACTCACCCGTCAGTCTATAAATACTTAGCCCCTCCCTCATTGTTAAGGGAGCAAAATCTCAGAGAGATAGTCCTAGAGAGAGAAAGAGAGCAAGTAGCCTAGAAGTAGTCAAGGCGGCGAAGTATTCAGGCAGGTGGCCAGGAAGAAGAAAAGCCAAGACGACGAAAACAGGTAAGAGCTAAGCTAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATAATTTCTACTAAGTGTAGATCACTGGCATCTGTAGCTTCAGTAGTAATTTCTACTAAGTGTAGATGCTCTTCCACGGAGACCTTCTCCATAATTTCTACTAAGTGTAGATTCTTGAGACCGTAGAGAGAGAGATAATTTCTACTAAGTGTAGATTTGCACAACAACTACCTGCATGCTAATTTCTACTAAGTGTAGATATGCCTGGGCACACTGCCGGGCATTAATTTCTACTAAGTGTAGATCGGTGGTCACTGCAGGGTTGCCGTTAATTTCTACTAAGTGTAGATAGAAGATGGAGAACTACCAGGGGTAATTTCTACTAAGTGTAGATCTTGAGAGGATGGACAACTACCATAATTTCTACTAAGTGTAGATGCTTAAGCAGGATGGAGCACCAGTAATTTCTACTAAGTGTAGATAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTA

crRNA array for LEA_Dehydrin in LbCpf1 SSTU system.

> tRANGly-DR-g5070-DR-g4487-DR-g4528(1)-DR-g4528(2)-DR-g2657-DR-g2675

-DR-g2676-DR-g2678-DR-g2679-DR-tRANGly

GGCTGGAGTACGCCCAGACCAGCGTGAAGCACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAAGGATCCGGTACCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATAATTTCTACTAAGTGTAGATCACTGGCATCTGTAGCTTCAGTAGTAATTTCTACTAAGTGTAGATGCTCTTCCACGGAGACCTTCTCCATAATTTCTACTAAGTGTAGATTCTTGAGACCGTAGAGAGAGAGATAATTTCTACTAAGTGTAGATTTGCACAACAACTACCTGCATGCTAATTTCTACTAAGTGTAGATATGCCTGGGCACACTGCCGGGCATTAATTTCTACTAAGTGTAGATCGGTGGTCACTGCAGGGTTGCCGTTAATTTCTACTAAGTGTAGATAGAAGATGGAGAACTACCAGGGGTAATTTCTACTAAGTGTAGATCTTGAGAGGATGGACAACTACCATAATTTCTACTAAGTGTAGATGCTTAAGCAGGATGGAGCACCAGTAATTTCTACTAAGTGTAGATAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGGTACCGGATCCTGATTGATCGATAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATAT

Note: NLS and stop coden TAA of the LbCpf1 expression cassette are marked in pink and gray, respectively. Sequences before NLS belong to the CDS of LbCpf1. The Bam HI restriction sites for crRNA array cloning are marked in italic sequences. Characters in purple are partial sequences of the NOS terminator.

Multiple sgRNA expression cassettes for targets 230, 250 and 240 in Cas9 TCTU-3 system.

> OsU6-guide230-gRNA-poly T-OsU31-guide250-gRNA-poly T-OsU62-guide240-

gRNA-poly T.

GGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGTACTCCACTTCTGTAACCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTAGAGCTCCAAGTTCTAGGATTTTCAGAACTGCAACTTATTTTATCAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGATGTGAAGAAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGATTTAAAAGAGTTGTGCAGATGATCCGTGGCACAACAATGGCGAAAACAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTAGAGTTTTGTGAAAGTTGAATTACGGCATAGCCGAAGGAATAACAGAATCGTTTCACACTTTCGTAACAAAGGTCTTCTTATCATGTTTCAGACGATGGAGGCAAGGCTGATCAAAGTGATCAAGCACATAAACGCATTTTTTTACCATGTTTCACTCCATAAGCGTCTGAGATTATCACAAGTCACGTCTAGTAGTTTGATGGTACACTAGTGACAATCAGTTCGTGCAGACAGAGCTCATACTTGACTACTTGAGCGATTACAGGCGAAAGTGTGAAACGCATGTGATGTGGGCTGGGAGGAGGAGAATATATACTAATGGGCCGTATCCTGATTTGGGCTGCGTCGGAAGGTGCAGCCCACGCGCGCCGTACCGCGCGGGTGGCGCTGCTACCCACTTTAGTCCGTTGGATGGGGATCCGATGGTTTGCGCGGTGGCGTTGCGGGGGATGTTTAGTACCACATCGGAAACCGAAAGACGATGGAACCAGCTTATAAACCCGCGCGCTGTAGTCAGCTTGCTGAAGCGGAGGCTGTTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT

Single sgRNA expression cassette for target 260 in Cas9 SSTU system

> OsU6-guide260-gRNA-poly T.

GGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGCATGCCATTGCTCCTGCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT

sgRNA array for targets 230, 250 and 240 in Cas9 SSTU system

> guide230-gRNA-linker-guide250-gRNA-linker-guide240-gRNA.

GTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAAGGATCCCTCGAGGGTACCGTACTCCACTTCTGTAACCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTGGTCTCGCAACAATGGCGAAAACAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTGGTCTCGCTGAAGCGGAGGCTGTTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTCTCGAGGGTACCGGATCCTGATTGATCGATAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATAT

Note: NLS and stop coden TAA of the Cas9 expression cassette are marked in green and gray, respectively. Sequences before NLS belong to the CDS of Cas9. The linkers are marked in bold and the Bam HI restriction sites for sgRNA array cloning are marked in italic sequences. Characters in purple are partial sequences of the NOS terminator.

Supplemental References

Bae S, Park J and Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473-1475

Čermák T, Curtin SJ, Gil-Humanes J,  Čegan R, Kono TJY, Konečná E, Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF (2017) A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196-1217

Feng Z, Zhang B, Ding W, Liu X, Yang D, Wei P, Cao F, Zhu S, Zhang F, Mao Y and Zhu J (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23:1229-1232

Gao Y, Zhao Y (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 56: 343-349

Liu W, Xie X, Ma X, Li J, Chen J and Liu YG (2015) DSDecode: A web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol Plant 8:1431-1433.

Ma X, Chen L, Zhu Q, Chen Y and Liu YG (2015) Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products. Mol Plant 8:1285-1287.

Nishimura A, Aichi I and Matsuoka M (2006) A protocol for Agrobacterium-mediated transformation in rice. Nat protocol 1:2796-2802

Wang M, Liu Y, Zhang C, Liu J, Liu X, Wang L, Wang W, Chen H, Wei C, Ye X, Li X and Tu J (2015) Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations. PloS One 10: e122755

Wang M, Mao Y, Lu Y, Tao X and Zhu JK (2017a) Multiplex gene editing in rice using the CRISPR-Cpf1 system. Mol Plant 10:1011-1013

Wang M, Lu Y, Botella JR, Mao Y, Hua K, Zhu JK (2017b) Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 System. Mol Plant 10:1007-1010

Wang XS, Zhu HB, Jin GL, Liu HL, Wu WR and Zhu J (2007) Genome-scale identification and analysis of LEA genes in rice (Oryza sativa L.). Plant Science 172: 414-420

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