1

Supplemental Information

Multiplex gene editing in rice using the CRISPR-Cpf1 system

Mugui Wang et al

Contents

Supplemental Figure 1. T-DNA constructs of FnCpf1 and LbCpf1 for single

gene editing in rice.

Supplemental Figure 2. The phenotypes of rice wild type (WT), OsPDS

chimera (Chi) and bi-allele (Bi) T0 mutants derived from Cpf1 gene editing.

Supplemental Figure 3. The genotype of OsRLK family in rice T0 transgenic

line #5 derived from FnCpf1-Multiplex gene editing.

Supplemental Figure 4. The genotype of OsRLK family in rice T0 transgenic

line #12 derived from FnCpf1-Multiplex gene editing.

Supplemental Figure 5. The genotype of OsRLK family in rice T0 transgenic

line #31 derived from FnCpf1-Multiplex gene editing.

Supplemental Figure 6. The genotype of OsBEL family in rice T0 transgenic

line #10 derived from LbCpf1-Multiplex gene editing.

Supplemental Figure 7. The genotype of OsBEL family in rice T0 transgenic

line #13 derived from LbCpf1-Multiplex gene editing.

Supplemental Figure 8. The genotype of OsBEL family in rice T0 transgenic

line #18 derived from LbCpf1-Multiplex gene editing.

Supplemental Figure 9. T-DNA constructs of FnCpf1 and LbCpf1 with

interchanged DR sequence.

Supplemental Table 1. Summary of the target sequences and mutations in

rice T0 plants transformed with the FnCpf1 and LbCpf1 single gene editing

systems.

Supplemental Table 2. Summary of the gene editing efficiency in rice T0

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plants transformed with FnCpf1 and LbCpf1 single gene editing systems with

interchanged DR sequences.

Supplemental Table 3. Primers used to amplify and sequence the target loci

in rice plants.

Supplemental Table 4. Potential off-target sites for FnCpf1-Multiplex gene

editing in rice OsRLK family.

Supplemental Table 5. Potential off-target sites for LbCpf1-Multiplex gene

editing in rice CYP81A family.

Supplemental Table 6. Primers used for off-target detection in rice plants.

Materials and methods

Supplemental Sequences. crRNA expression cassette of FnCpf1 and LbCpf1

multiplex gene editing system.

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LB RB

OsU6 LbCpf1

LbCpf1 cassette

NOSUbiguide DR

NLSNLS3XFlagPoly T

CrRNA cassette

LB RB

OsU6 FnCpf1

FnCpf1 cassette

NOSUbiguide DR

NLSNLS3XFlagPoly T

CrRNA cassette

A

B

LB RB

OsU6 LbCpf1

LbCpf1 cassette

NOSUbiguide DR

NLSNLS3XFlagPoly T

CrRNA cassette

LB RB

OsU6 FnCpf1

FnCpf1 cassette

NOSUbiguide DR

NLSNLS3XFlagPoly T

CrRNA cassette

A

B

Supplemental Figure 1. T-DNA constructs of FnCpf1 and LbCpf1 for single gene editing in rice. (A) The construct of FnCpf1 contains FnCpf1 expression cassette and crRNA expression cassette. FnCpf1 was inserted downstream of the ZmUbi promoter. A NOS terminator was placed at the end of FnCpf1 ORF. The SV40 derived nuclear localization signal (NLS) was fused translationally to both N and C-termini of FnCpf1. A 3×Flag was in-frame fused to the N-terminus of NLS. The crRNA expression cassette includes a mature DR and 22-24 bp of guide sequence, under the control of OsU6 promoter and terminates by 7 bp polyT sequence. (B) The construct of LbCpf1 for single gene editing. The included elements are similar to those of FnCpf1.

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Supplemental Figure 2. The phenotypes of rice wild type (WT), OsPDS chimera (Chi) and bi-allele (Bi) T0 mutants derived from Cpf1 gene editing. Bar equals 1 cm.

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Osrlk-798 (Bi) 5’-TTTCCATTCCCCATCCCGTTCTTTGGCCCCTTCACGAGCCCACAAGAC (Reference) 5’-TTTCCATTCCCCATCCCGT-------CCCCTTCACGAGCCCACAAGAC (-7) 5’-TTTCCATTCCCCATCCCGT-------------CACGAGCCCACAAGAC (-13)

Osrlk-799 (Bi) 5’-TTTGGAAGGACAGTTAGGCAGCCTGGGGAACTTCACTTCAATAACCAC (Reference) 5’-TTTGGAAGGACAGTTAGG-------GGGAACTTCACTTCAATAACCAC (-7) 5’-TTTGGAAGGACAGTTAGGCAGC-------------------------- (-106)

Osrlk-802 (Bi) 5’-TTTCGCAAGAGCGCCTCATCCACGCTTGTGAGCCTCGAGTACTCGAAT (Reference) 5’-TTTCGCAAGAGCGCCTCA------------AGCCTCGAGTACTCGAAT (-12) 5’-TTTCGCAAGAGCGCCTCATCC------GTGAGCCTCGAGTACTCGAAT (-6)

Osrlk-803 (Bi) 5’-TTTACCTCGACGTCAACCACCTGACCGGGCCACTGCCCGTGGAGATTG (Reference) 5’-TTTACCTCGACGT---------------------------GGAGATTG (-27) 5’-TTTACCTCGACGTCAACCA--------------------TGGAGATTG (-20)

Supplemental Figure 3. The genotype of OsRLK family in rice T0 transgenic line #5 derived from FnCpf1-Multiplex gene editing. The genotypes, sequences and original sequencing chromatograms for each target site are shown. Each dashed line represents a deleted nucleotide. The number after ‘-’ represents the number of bases that have been deleted. PAM-guide sequence is marked in grey and the PAM motif (TTN) is marked in bold (sequence text) or underlined (chromatogram). Bi: possible bi-allele.

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Osrlk-798 (Ho) 5’-TTTCCATTCCCCATCCCGTTCTTTGGCCCCTTCACGAGCCCACAAGAC (Reference) 5’-TTTCCATTCCCC------------------TTCACGAGCCCACAAGAC (-18) 5’-TTTCCATTCCCC------------------TTCACGAGCCCACAAGAC (-18)

Osrlk-799 (He) 5’-TTCAATGCTGCAAATTTGGAAGGACAGTTAGGCAGCCTGGGGAACTTC (Reference) 5’-TTCAATGCTGCAAATTTGGAAGGACAGTTAGGCAGCCTGGGGAACTTC (WT) 5’-TTCAAT-----------------(-50)---------------------AACCACTATGT

Osrlk-802 (Bi) 5’-TTTCGCAAGAGCGCCTCATCCACGCTTGTGAGCCTCGAGTACTCGAAT (Reference) 5’-TTTCGC---------------------------CTCGAGTACTCGAAT (-27) 5’-TTTCGCAAGA------------------------TCGAGTACTCGAAT (-24)

Osrlk-803 (He) 5’-CGGAGCTCACCGACCTTTACCTCGACGTCAACCACCTGACCGGGCCAC (Reference) 5’-CGGAGCTCACCGACCTTTACCTCGACGTCAACCACCTGACCGGGCCAC (WT) 5’-CGGAGC------------------(-45)-------------------CCGTGGAGATTG

Supplemental Figure 4. The genotype of OsRLK family in rice T0 transgenic line #12 derived from FnCpf1-Multiplex gene editing. The genotypes, sequences and original sequencing chromatograms for each target site are shown. Each dashed line represents a deleted nucleotide. The number after ‘-’ represents the number of bases that have been deleted. PAM-guide sequence is marked in grey and the PAM motif (TTN) is marked in bold (sequence text) or underlined (chromatogram). WT: wild-type, He: possible heterozygote, Ho: possible homozygote, Bi: possible bi-allele.

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Osrlk-798 (Bi) 5’-TTTCCATTCCCCATCCCGTTCTTTGGCCCCTTCACGAGCCCACAAGAC (Reference) 5’-TTTCCATTCCCCATCCCG--------CCCCTTCACGAGCCCACAAGAC (-8) 5’-TTTCCATTCCCCATCCCGTT------CCCCTTCACGAGCCCACAAGAC (-6)

Osrlk-799 (He) 5’-CGAGTGTTTATATTTTGTGCTTTTATCAACTCATTGTTTGTAGAATTTTCAATGCTGCAAATTTGGAAGGACAGTTAGGCAGCCTGGGGAACTT (Reference) 5’-CGAGTGTTTATATTTTGTGCTTTTATCAACTCATTGTTTGTAGAATTTTCAATGCTGCAAATTTGGAAGGACAGTTAGGCAGCCTGGGGAACTT (WT) 5’-CGAGTGT---------------------(-80)------------------------------------------------------GGAACTT

Osrlk-802 (Bi) 5’-TTTCGCAAGAGCGCCTCATCCACGCTTGTGAGCCTCGAGTACTCGAAT (Reference) 5’-TTTCGCAAGAGCGCCTCAT----------GAGCCTCGAGTACTCGAAT (-10) 5’-TTTCGCAAGAGCGCCTCATCCA------TGAGCCTCGAGTACTCGAAT (-6)

Osrlk-803 (He) 5’-TTTACCTCGACGTCAACCACCTGACCGGGCCACTGCCCGTGGAGATTG (Reference) 5’-TTTACCTCGACGTCAACCACCTGACCGGGCCACTGCCCGTGGAGATTG (WT) 5’-TTTACCTCGACGTCAAC----------GGCCACTGCCCGTGGAGATTG (-10)

Supplemental Figure 5. The genotype of OsRLK family in rice T0 transgenic line #31 derived from FnCpf1-Multiplex gene editing. The genotypes, sequences and original sequencing chromatograms for each target site are shown. Each dashed line represents a deleted nucleotide. The number after ‘-’ represents the number of bases that have been deleted. PAM-guide sequence is marked in grey and the PAM motif (TTN) is marked in bold (sequence text) or underlined (chromatogram). WT: wild-type, He: possible heterozygote, Bi: possible bi-allele.

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Osbel-230 (He) 5’-TTTGCTCGGTGAGTACAAGTCTAAAAGTCTGCTCTCCGATGGATCGGG (Reference) 5’-TTTGCTCGGTGAGTACAAGTCTAAAAGTCTGCTCTCCGATGGATCGGG (WT) 5’-TTTGCTCGGTGA----------------CTGCTCTCCGATGGATCGGG (-16)

Osbel-240 (He) 5’-TTTCATCTCCTTCTAGAAGCACAAGCGCCGCTCGGTATAAAGGCAGAC (Reference) 5’-TTTCATCTCCTTCTAGAAGCACAAGCGCCGCTCGGTATAAAGGCAGAC (WI) 5’-TTTCATCTCCTTCTAGAA--------GCCGCTCGGTATAAAGGCAGAC (-8)

Osbel-250 (He) 5’-AAGACTGAGCCAGAGGTCTACAAGGATATAGTGATCGTGAATCTTTGTGCGGTGAGTGAGTACGTACTTTCGCCAGGTTTCAAATCATCTC (Reference) 5’-AAGACTGAGCCAGAGGTCTACAAGGATATAGTGATCGTGAATCTTTGTGCGGTGAGTGAGTACGTACTTTCGCCAGGTTTCAAATCATCTC (WT) 5’-AAGACTGAGCCAG---------------------------------------------------------------GTTTCAAATCATCTC (-63)

Osbel-260 (Bi) 5’-TTTGGTGCCGGAACAGAGACTACATCAACCACAATAGAGTGGGCGATG (Reference) 5’-TTTGGTGCCGGAACAGA-----------CCACAATAGAGTGGGCGATG (-11) 5’-TTTGGTGCCGGAACAGAG----------CCACAATAAAGTGGGCGATG (-10/S1)

Supplemental Figure 6. The genotype of OsBEL family in rice T0 transgenic line #10 derived from LbCpf1-Multiplex gene editing. The nucleotide in red represents substitution (s). -n/sn means simultaneous nucleotide deletion/substitution of the indicated number at the site. PAM-guide sequence is marked in grey and the PAM motif (TTTN) is marked in bold (sequence text) or underlined (chromatogram). The PCR amplicons of Osbel-240 target site was sequenced with reverse primer so that the PAM is not marked in chromatogram. Other information is as in Supplemental Figure 5.

Reverse sequencing

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Osbel-230 (Bi) 5’-TTTGCTCGGTGAGTACAAGTCTAAAAGTCTGCTCTCCGATGGATCGGG (Reference) 5’-TTTGCTCGGTGAGTA--------------TGCTCTCCGATGGATCGGG (-14) 5’-TTTGCTCGGTGAGTA------------------CTCCGATGGATCGGG (-18)

Osbel-240 (Bi) 5’-TTTCATCTCCTTCTAGAAGCACAAGCGCCGCTCGGTATAAAGGCAGAC (Reference) 5’-TTTCATCTCCTTCTAGAAGC------GCCGCTCGGTATAAAGGCAGAC (-6) 5’-TTTCATCTCCTTCTAGAA--------GCCGCTCGGTATAAAGGCAGAC (-8)

Osbel-250 (He) 5’-TTTGTGCGGTGAGTGAGTACGTACTTTCGCCAGGTTTCAAATCATCTC (Reference) 5’-TTTGTGCGGTGAGTGAGTACGTACTTTCGCCAGGTTTCAAATCATCTC (WT) 5’-TTTGTGCGGTGAGTGAGTA-------TCGCCAGGTTTCAAATCATCTC (-7)

Osbel-260 (Bi) 5’-TTTGGTGCCGGAACAGAGACTACATCAACCACAATAGAGTGGGCGATG (Reference) 5’-TTTGGTGCCGGAACAGAG--------AACCACAATAGAGTGGGCGATG (-8) 5’-TTTGGTGCCGGAACAGAG-----------------------GGCGATG (-23)

Supplemental Figure 7. The genotype of OsBEL family in rice T0 transgenic line #13 derived from LbCpf1-Multiplex gene editing. The genotypes, sequences and original sequencing chromatograms for each target site are shown. Each dashed line represents a deleted nucleotide. The number after ‘-’ represents the number of bases that have been deleted. PAM-guide sequence is marked in grey and the PAM motif (TTTN) is marked in bold (sequence text) or underlined (chromatogram). The PCR amplicons of Osbel-240 target site was sequenced with reverse primer so that the PAM is not marked in chromatogram. WT: wild-type, He: possible heterozygote, Bi: possible bi-allele.

Reverse sequencing

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Osbel-230 (Bi) 5’-TTTGCTCGGTGAGTACAAGTCTAAAAGTCTGCTCTCCGATGGATCGGG (Reference) 5’-TTTGCTCGGTGAGTAC----------GTCTGCTCTCCGATGGATCGGG (-10) 5’-TTTGCTCGGTGAGTACAA------------GCTCTCCGATGGATCGGG (-12)

Osbel-240 (Ho) 5’-TTTCATCTCCTTCTAGAAGCACAAGCGCCGCTCGGTATAAAGGCAGAC (Reference) 5’-TTTCATCTCCTTCTAG------AAGCGCCGCTCGGTATAAAGGCAGAC (-6) 5’-TTTCATCTCCTTCTAG------AAGCGCCGCTCGGTATAAAGGCAGAC (-6)

Osbel-250 (Bi) 5’-TTTGTGCGGTGAGTGAGTACGTACTTTCGCCAGGTTTCAAATCATCTC (Reference) 5’-TTTGTGCGGTGAGTG----------TTCGCCAGGTTTCAAATCATCTC (-10) 5’-TTTGTGCGGTGAGTGAGT---TACTTTCGCCAGGTTTCAAATCATCTC (-3)

Osbel-260 (Bi) 5’-TTTGGTGCCGGAACAGAGACTACATCAACCACAATAGAGTGGGCGATG (Reference) 5’-TTTGGTGCCG---------------------------AGTGGGCGATG (-27) 5’-TTTGGTGCCGGAACAGAGAA------AACCACAATAGAGTGGGCGATG (-6/S1)

Supplemental Figure 8. The genotype of OsBEL family in rice T0 transgenic line #18 derived from LbCpf1-Multiplex gene editing. The genotypes, sequences and original sequencing chromatograms for each target site are shown. Each dashed line represents a deleted nucleotide. The number after ‘-’ represents the number of bases that have been deleted. The nucleotide in red represents substitution (s). -n/sn means simultaneous nucleotide deletion/substitution of the indicated number at the site. PAM-guide sequence is marked in grey and the PAM motif (TTTN) is marked in bold (sequence text) or underlined (chromatogram). Ho: possible homozygote, Bi: possible bi-allele.

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LB RB

OsU6 FnCpf1

FnCpf1 cassette

NOSUbiguide LbDR

NLSNLS3XFlagPoly T

Lb-CrRNA cassette

LB RB

OsU6 LbCpf1

LbCpf1 cassette

NOSUbiguide FnDR

NLSNLS3XFlagPoly T

Fn-CrRNA cassette

A

B

LB RB

OsU6 FnCpf1

FnCpf1 cassette

NOSUbiguide LbDR

NLSNLS3XFlagPoly T

Lb-CrRNA cassette

LB RB

OsU6 LbCpf1

LbCpf1 cassette

NOSUbiguide FnDR

NLSNLS3XFlagPoly T

Fn-CrRNA cassette

A

B

Supplemental Figure 9. T-DNA constructs of FnCpf1 and LbCpf1 with interchanged DR sequence. (A) The construct of FnCpf1 with mature DR sequence from LbCpf1. (B) The construct of LbCpf1 with mature DR sequence from FnCpf1. The included elements are similar to those in Supplemental Figure 1.

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Supplemental Table 1. Summary of the target sequences and mutations in rice T0 plants transformed with the FnCpf1 and LbCpf1 single gene editing systems.

Note: Bi: possible bi-allele, He: possible heterozygote, Chi: Chimera, WT: Wild Type. The PAM motif

(TTN for FnCpf1 and TTTN for LbCpf1) is marked in bold.

Supplemental Table 2. Summary of the gene editing efficiency in rice T0 plants transformed with FnCpf1 and LbCpf1 single gene editing systems with interchanged DR sequences.

Cpf1 DR Target loci Plantlets

identify Mutant

Mutation

rate % Genotype

FnCpf1 LbDR OsPDS-1 30 3 10 1He+2Chi+27WT

LbCpf1 FnDR OsPDS-1 30 11 36.7 1Bi+3He+7Chi+19WT

FnCpf1 LbDR OsBEL-2 37 8 21.6 4He+4Chi+29WT

LbCpf1 FnDR OsBEL-2 42 9 21.4 3He+6Chi+33WT

Note: Bi: possible bi-allele, He: possible heterozygote, Chi: Chimera, WT: Wild Type.

Target

loci PAM-guide sequence (5’-3’)

Plantlets

identify Mutant

Mutation

rate % Genotype

FnCpf1

OsEPSPS-1 TTTCCTGTTGAGAAGGATGCGAAAGAG 24 0 0 24WT

OsEPSPS-2 TTTCCACCAGCAGCAGTCACGGCTGC 24 4 16.7 2He+2Chi+20WT

OsBEL-1 TTTCATCTCCTTCTAGAAGCACAAGCG 36 7 19.4 6He+1Chi+19WT

OsBEL-2 TTTAAGCAGGTCGTCGACGAGATCATC 24 3 12.5 2He+1Chi+21WT

OsPDS-1 TTTTGCGGGACAACTTCCTACTCATAG 24 2 8.3 1He+1Chi+22WT

OsPDS-2 TTTGCTCCTGCAGAGGAATGGGTTGGA 26 0 0 26WT

LbCpf1

OsEPSPS-1 TTTCCTGTTGAGAAGGATGCGAAAGAG 36 2 5.6 1He+1Chi+34WT

OsEPSPS-2 TTTCCACCAGCAGCAGTCACGGCTGC 24 5 20.8 4He+1Chi+19WT

OsBEL-1 TTTCATCTCCTTCTAGAAGCACAAGCG 24 15 62.5 2Bi+6He+7Chi+9WT

OsBEL-2 TTTAAGCAGGTCGTCGACGAGATCATC 35 7 20 4He+3Chi+28WT

OsPDS-1 TTTTGCGGGACAACTTCCTACTCATAG 34 25 73.5 7Bi+8He+10Chi+9WT

OsPDS-2 TTTGCTCCTGCAGAGGAATGGGTTGGA 56 1 1.8 1He+55WT

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Supplemental Table 3. Primers used to amplify and sequence the target loci in rice plants.

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

OsEPSPS-1 CAACTTTGGAGGTTTCGCACTG TCGCTTGAGCTTGGCAGGAATA 1023

OsEPSPS-2 CAACTTTGGAGGTTTCGCACTG TCGCTTGAGCTTGGCAGGAATA 1023

OsBEL-1 GCAACGGAGTGAGTAGAAGT GGACGCGGCCGCGGCGCGGTACATCCT 765

OsBEL-2 CAGCTGCTGGTCTCGTTCAA TGCTGAATGCTGATAGTGCC 806

OsPDS-1 GAAACTCGGAAGATTAGGGA AAGACCACGATGTGACTGCT 846

OsPDS-2 GTGTCTCTTCTAATTCCTCATCAGT AGCGTTTACCTCCACTACAGACT 1045

OsRLK-798 TGTTCTCAGTTCTCACCACCAT GTCTCACTCAGCTTTCCTCC 581

OsRLK-799 GGTGAAATGCTGGGTTCTGT CAGCAAGGAACATAAGGCATCTC 798

OsRLK-802 GCCATTGCACTGGAACTCAC ATCCCCTCAGGCCAACAATG 546

OsRLK-803 CTGATGGCGCTCAAGGAGAG CTCACCTTGGAGATTCTCCATG 345

OsBEL-230 GGAAGCCCAGGAGTACAAGC CTTCTCGCGTTACCCGCTGT 438

OsBEL-240 GCCTTCCAGGTACGTAGCAAT AGTGGAGCAAGAAGAGGATAGC 598

OsBEL-250 AGGCCCAGGAGTTCAAGAAT AGCGTTCAAAATTCATCAATAGTC 452

OsBEL-260 GAGCATGATTTCCGTGCTGC ACGTTGACAAGCAACATCGC 555

Supplemental Table 4. Potential off-target sites for FnCpf1-Multiplex gene editing in rice OsRLK family.

Target Off-target No. PAM-guide sequence (5’-3’) Chr. Position Direction Mismatches

(nt)

Bulge

Size

On-Target TTNTCCATTCCCCATCCCGTTCTTTGGC

OFF-1 TTATCCATTCCAtCATCCCtTTCTcTctC chr10 864400 + 5 1

OFF-2 TTATCCATTCCCCtTCtCtTTCTcTctC chr3 14357761 + 6 0

OsRLK

-798

OFF-3 TTTTCCATTCCCCgTgCCcTTCcTTatC chr1 40797257 - 6 0

On-Target TTNTGGAAGGACAGTTAGGCAGCCTGGG

OFF-1 TTGTGGAAGGctAGcTAGGCccCaTGGG chr1 8128271 + 6 0

OFF-2 TTGTGGAAGGACAGTTCAGaCgGgCTctG chr5 28259975 + 5 1

OsRLK

-799

OFF-3 TTGTGGAAGaACAGTTATGGCAGCtgGct chr5 13760575 - 5 1

On-Target TTNTCGCAAGAGCGCCTCATCCACGCTT

OFF-1 TTCTCGCAAGAGgGCtTCcTCCAgGtTT chr3 7144486 + 5 0

OFF-2 TTTTCtCAAGctCGCgTCAaCCACGCgT chr12 14506460 + 6 0

OsRLK

-802

OFF-3 TTTTgGaAAGAGCGCaaCAcCCACGTCTT chr9 3229830 + 5 1

On-Target TTNTACCTCGACGTCAACCACCTGACCG

OFF-1 TTCTACCTCGcCaTggACCACaTGtCCG chr7 465960 + 6 0

OFF-2 TTCTACCTCaACGTCACgCCgCgcGACCG chr10 21077997 - 5 1

OsRLK

-803

OFF-3 TTCTACCTCGACGgatACtACtcGtCCG chr1 28223697 + 7 0

Note: the mismatches are labeled in lowercase and the PAM motif (TTN) is marked in bold.

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Supplemental Table 5. Potential off-target sites for LbCpf1-Multiplex gene editing in rice CYP81A family.

Target Off-target No. PAM-guide sequence (5’-3’) Chr. Position Direction Mismatches

(nt)

Bulge

Size

On-Target TTTNCTCGGTGAGTACAAGTCTAAAAG

OFF-1 TTTTCTCcGTttGTACAAaTCTtAAAG chr7 19080726 - 5 0

OFF-2 TTTTCTCGGTGAtcAgAAGTtggAAAG chr2 10887403 + 6 0

OsBEL

-230

OFF-3 TTTGCTCGGTGAGTAgAAGTaTtAAgc chr10 4994993 + 5 0

On-Target TTTNATCTCCTTCTAGAAGCACAAGCG

OFF-1 TTTCATCTCCTcCcAGAAGCAtAtGac chr8 5309581 - 6 0

OFF-2 TTTGATCTCCTgCgAGAAGaAgAAGac chr3 20835040 - 6 0

OsBEL

-240

OFF-3 TTTCATCTCCTcCgAGAAGCgCAgcaG chr6 16937851 + 6 0

On-Target TTTNTGCGGTGAGTGAGTACGTACTTTC

OFF-1 TTTCTtCGGTGAacGAGTACaTAaTTTC chr12 23178070 - 5

OFF-2 TTTTTGCGGgGAGTacGTgCGTgCTcTC chr1 33045431 + 6

OsBEL

-250

OFF-3 TTTATGgGaCTGAGTGAGTACGTACTTTC chr1 2952468 - 2 1

On-Target TTTNGTGCCGGAACAGAGACTACATCA

OFF-1 TTTGcTGCtGGAACAGAGACTACAgCA chr3 31389410 + 3

OFF-2 TTTCGTGCCGGAAgAtAcgCTAaATtA chr4 19465256 + 6

OsBEL

-260

OFF-3 TTTCGCTGCtGGctCAGAGACTACATCt chr1 42534623 + 4 1

Note: the mismatches are labeled in lowercase and the PAM motif (TTTN) is marked in bold.

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Supplemental Table 6. Primers used for off-target detection in rice plants.

Target OFF-target

No. Forward primer (5' to 3') Reverse primer (5' to 3')

PCR product

(bp)

OFF-1 TTTCCCCACTATTTTCCGGTCA TATGGAGATGACGCTGTGGC 484

OFF-2 GTCGTCCCGACATAAGCACA GGGTGTTGCCATACCTCACA 567 OsRLK

-798 OFF-3 TCGTTGTCGATCGCCTGAAT GTGCGTGAGAGAGAATCTAAGAC 435

OFF-1 CCGGTTTACAACCCAGGACT GCACAGGGGTCTTCTAGAGC 589

OFF-2 GTACGCTCGCGGGATATGTT AGTGCCAGGATTAGGCTGTT 697 OsRLK

-799 OFF-3 TCTAAGCAAGGGTCCATGCAG CACTATCTGGTGCGAGTCGT 691

OFF-1 CATCGCATGGGCACTACACT TCTGTCAGCAGCAGTTCCAT 693

OFF-2 CCCCTATTCTTCCGGCAACT GCTTCGGTGAGACTGGTGACTT 665 OsRLK

-802 OFF-3 GCACAAGCGCCACCAAAGTA TAACGCAGAAACACCGAGCA 559

OFF-1 AGCCCATCTGTTTGGATTCTTGA CACCATCGTGTGCGAGTGC 785

OFF-2 GTGGTCACCTCAACGTCCAC CGACGCGGCCTCTCTAGTA 683 OsRLK

-803 OFF-3 GGGCGCAAGGTATCGAACT AGAAGGGCTCGAAGTCGG 700

OFF-1 TGACAAAAATAAGGATAGCGTTGTG TGCTGTTGGCTATGAAGGACA 781

OFF-2 ACTGGCTATTCGGTGGGAGA GGTCGTACAGCGTGTGAACT 740 OsBEL

-230 OFF-3 TGTGCTACCTAGAGTGCCAT CTGGTTCACCTGCACCGAAT 552

OFF-1 AGGTGTCAGCTCTGTGCATC GGCCCTACACAATTAAATCCGC 655

OFF-2 TTGACGGGAGTCTCCTTCTC CTTCACCGAAACACTGGTAT 768

OsBEL

-240 OFF-3 TGTTCCCCAACTACCGCTTG GGGCCAGGCTTTTTCTGATG 586

OFF-1 GTTCGGCACTGTTAGTATCCA CATATCCGTATTTGAGTCGGT 568

OFF-2 AACTGCGTAGAACCCCTGC GCCGAGGTCTGCCTGATTTA 563

OsBEL

-250 OFF-3 GACATGGTGCTCATCAACGA CCCGGATGTACCTCCAAGAA 749

OFF-1 TGCTTCAACGAAGTTACAATCAC TCTGCGGATGATTCGTGAGG 575

OFF-2 CCCGATTGCGAAAGCATGG GCCTTCTTCGGGTCCCTTATC 524 OsBEL

-260 OFF-3 ACTTGTGTCGTGCTATGTGGT CGGGCCTAAACTCATTTGCG 569

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Materials and methods

Vector construction

The human codon optimized Cpf1 from Francisella novicida (FnCpf1, pY004)

and Lachnospiraceae bacterium ND2006 (LbCpf1, pY016) were obtained from

Addgene. The coding sequences of FnCpf1 and LbCpf1 were cloned from

pY004 and pY016, respectively, to replace the Cas9 coding sequence in the

OsU6-Cas9 plasmid described previously (Feng et al., 2013; Zhang et al.,

2014). For single gene editing, the mature DR sequence of FnCpf1 and

LbCpf1 were synthesized plus two reversely connected BsaI sites for cloning

the designed targeting sequences. A 7bp polyT short sequence was set to

terminate the crRNA (OsU6-DR-guide) expression cassette. The oligos (22-24

nt) for each target were annealed and cloned into the above BsaI sites as

described previously (Feng et al., 2013; Zhang et al., 2014). For multiplex

gene editing, the short DR-guide array that includes several mature DRs and

22-24 bp target sequences was directly synthesized by Sangon Biotech

(Shanghai, China) and cloned into the crRNA expression cassette

(Supplemental Sequences).

Rice transformation procedure and growth conditions

The constructs were introduced into A. 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 performed as described previously

(Nishimura et al., 2007; Wang et al., 2015). Three days after transformation,

the calli were transferred onto N6 medium supplemented with 2 mg/L

2,4-dichlorophenoxyacetic acid, 2 g/L casamino acids, 3 g/L gelrite, 30 g/L

sucrose and 40 mg/L hygromycin for 14 days. The resulting resistant calli were

selected and transferred onto N6 medium supplemented with 4 mg/L

6-benzylaminopurine, 1 mg/L α-naphthaleneacetic acid, 30 g/L sucrose, 5 g/L

gelrite and 25 mg/L hygromycin for regenerating plantlets. The subsequent

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plantlets were transplanted in Yoshida’s culture solution (Yoshida et al., 1976)

for one week and then transplanted in soil in greenhouse under standard

growth conditions (12-h light at 28°C rotated with 12-h darkness at 22°C). The

average light intensity was 30000 Lux.

Genotyping and sequence analysis of targeted mutations

After the hygromycin-resistant transgenic plants were generated, genomic

DNA was isolated from the fresh leaves of each T0 transgenic plant. The target

loci were amplified by PCR and then the resulting products were analyzed by

Sanger sequencing. The sequencing chromatograms were analyzed by

DSDecode (Liu et al., 2015; Ma et al., 2015) and further confirmed by TA

cloning and sequencing, where 10-20 positive colonies for each sample were

sequenced. Mutation ratio was estimated by scoring the number of plants with

mutation around the target sequence relative to the total number of identified

plants. Primer sets for the PCR and sequencing are listed in Supplemental

Table 3.

Detection of off-target effects

Potential off-target sites were predicted using the online tool Cas-OFFinder

(Bae et al., 2014). Previous studies found that the off-target effect derived from

not only those containing Watson-Crick base-pairing mismatches to the guide,

but also insertion and deletion mis-matches in the guide-target heteroduplex

(Bae et al., 2014; Lin et al., 2014; Ran et al., 2015). We took into account of the

indel-mismatches to verify the off-target effect in this study. Homologous

sequences with mismatches and/or indels up to 7 bp with the target sequence

were listed. According to the number of mismatches and/or indels, and the

distance of these mismatches/indels from the seed region of Cpf1 crRNA,

three most likely off-target sites were further selected. The selected potential

off-target sites were examined by site-specific PCR in at least 5 mutant lines

with all four targets mutated and direct Sanger sequencing. The sequences of

the potential off-target sites and related PCR primers are listed in

Supplementary Tables 4-6.

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Supplemental sequences The crRNA expression cassette of FnCpf1 multiplex gene editing system >OsU6-DR-798guide-DR-799guide-DR-802guide-DR-803guide-poly T. GGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGTAATTTCTACTGTTGTAGATCATTCCCCATCCCGTTCTTTGGCTAATTTCTACTGTTGTAGATGAAGGACAGTTAGGCAGCCTGGGTAATTTCTACTGTTGTAGATGCAAGAGCGCCTCATCCACGCTTTAATTTCTACTGTTGTAGATCCTCGACGTCAACCACCTGACCGTTTTTTT

The crRNA expression cassette of LbCpf1 multiplex gene editing system > OsU6-DR-240guide-DR-260guide-DR-230guide-DR-250guide-poly T. GGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGTAATTTCTACTAAGTGTAGATATCTCCTTCTAGAAGCACAAGCGTAATTTCTACTAAGTGTAGATGTGCCGGAACAGAGACTACATCATAATTTCTACTAAGTGTAGATCTCGGTGAGTACAAGTCTAAAAGTAATTTCTACTAAGTGTAGATTGCGGTGAGTGAGTACGTACTTTCTTTTTTT

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searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473-1475.

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.

Lin Y., Cradick T.J., Brown M.T., Deshmukh H. and Ranjan P. et al. (2014). CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42:7473-7485.

Liu W., Xie X., Ma X., Li J., Chen J. and Liu Y.G. (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 Y.G. (2015). Rapid decoding of

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sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products. Mol. Plant 8:1285-1287.

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