SUPPLEMENTARY INFORMATION - Nature Research...DOI: 10.1038/NPLANTS.2016.139 SUPPLEENTAR INORATION 10 / 20 exon 2 deletion Supplementary Figure 8. Genotypes of the T 1 progeny from

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2016.139

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SUPPLEMENTARY MATERIALS

Table of Contents

Supplementary Figure 1. sgRNA-induced targeted mutations in the OsEPSPS gene

in rice protoplasts.

Supplementary Figure 2. Construction of the targeting vector pHUN411-C3C5 in

gene replacement.

Supplementary Figure 3. DNA sequence of the TIPS- E2 donor template.

Supplementary Figure 4. Outcome of PCR/RE assays to detect C3 and C5 sgRNA-

induced mutations in 14 T0 mutant plants.

Supplementary Figure 5. DNA sequence of the E2-TIPS-E8 donor template.

Supplementary Figure 6. Outcome of PCR/RE assays to detect C3 sgRNA-induced

mutations in 14 T0 mutant plants.

Supplementary Figure 7. Sequences of the ORFs of EPSPS from the TIPS mutants.

Supplementary Figure 8. Genotypes of the T1 progeny from the T0 plants with point

mutations and exon 2 deletion.

Supplementary Figure 9. The seed-setting rates of the T0 mutant plants.

Supplementary Figure 10. Intron-mediated gene replacement with GFP coding

sequence at OsDEP1 locus via NHEJ pathway in rice protoplasts.

Supplementary Table 1. CRISPR/Cas9 target loci and sequences.

Supplementary Table 2. Primers used and their applications.

Supplementary Table 3. Potential off-target sites of C3 and C5 detected in rice

protoplasts and TIPS T0 plants.

Supplementary Table 4. Genetic analysis of T0 plants with homozygous mutation at

C3 and C5 or C3 and their transmissions to the T1 generation.

Supplementary Table 5. Genetic analysis of intron targeting-mediated T0 mutations

in OsEPSPS and their transmissions to the T1 generation.

Supplementary Table 6. Analysis of the gene replacement, targeted gene insertion

and Cas9-free in the T1 generation.

Gene replacements and insertions in rice by intron targeting using CRISPR–Cas9

http://dx.doi.org/10.1038/nplants.2016.139

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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2016.139

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Supplementary Figure 1. sgRNA-induced targeted mutations in the OsEPSPS gene

in rice protoplasts. (a) sgRNAs targeting the intron 1 and 2 and exon 2 of OsEPSPS.

(b) Outcome of PCR/RE assays to detect sgRNA-induced mutations in OsEPSPS in

rice protoplasts. Primers were used to amplify fragments containing each of the

sgRNA target site individually. Lanes marked with “C1, C2, C3, C4 or C5” are from

digested sgRNA-transformed protoplasts; lanes marked with “2” and “3” are from

digested and undigested wild type controls. Red arrowheads indicate bands with

mutations. The numbers at the bottom of the gel indicate indel mutation frequencies

measured from the band intensities. The sequences of sgRNA-induced mutations in

the sgRNA sites in the protoplasts are shown on the right. The sgRNA target

sequences (C1, C2, C3, C4 and C5) are underlined and the PAM is in green. Indels

were detected at the expected positions. The numbers on the side indicate the type of

mutation and how many nucleotides are involved.


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Supplementary Figure 2. Construction of the targeting vector pHUN411-C3C5 in

gene replacement. (a) Cumulative data corresponding to the indel frequencies from

the C6 sgRNA driven by OsU3P and TaU3P in rice protoplasts, respectively (n=3). (b)

Schematic of the targeting vector pHUN411-C3C5. The C3 sgRNA (shown in green)

is driven by the OsU3 snRNA promoter and the C5 sgRNA (shown in purple) by the

TaU3 snRNA promoter. Hpt (shown in red) is driven by 2×35S promoter. Cas9

(shown in brown) is driven by the maize Ubiquitin promoter.




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ggtactaaatatacaatcccttgggtttatttgtttccaagcatgtcattaacttat

cttaatgtggacaagaaactgatgcctgcttacattgctattatttcaagcgggtat

tgatcctttgacatgtgattgatcatttttttttctctggttattagGGCACAACAG

TGGTGGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAG

CCCTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCT

GTGGTGGCAAGTTTCCTGTTGAGAAGGATGCGAAAGAGGAAGTGCAACTCTTCTTGG

GGAACGCTGGAATTGCAATGCGATCGTTGACAGCAGCCGTGACTGCTGCTGGTGGAA

ATGCAACgtatgtttttttttttaatgtttatgaaaatatgtatggaattcatggg

Supplementary Figure 3. DNA sequence of the TIPS-E2 donor template. The

sequences of the exon are in upper letters and the sequences of the introns are in lower

case letters. The sgRNA C3 and C5 site are marked in green and purple, respectively.

The DNA sequence of the conserved motif is underlined and the 3 nucleotide

substitutions are highlighted in red.




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Supplementary Figure 4. Outcome of PCR/RE assays to detect C3 and C5 sgRNA-

induced mutations. (a) Lanes T0-RP1 to T0-RP14, PCR fragments amplified from the

transgenic rice plants digested by BsaJI. The primers used for C3 site are EC3-F and

EC3-R. Lanes wt, PCR fragments amplified from a wild type control plant with and

without digestion. (b) PCR fragments amplified from the transgenic rice plants

digested by EcoRI. The primers used for C5 site are EPSPS-F and EPSPS-R. The

bands marked by red arrowheads are caused by sgRNA-induced mutations. (c)

Sequences of the two sgRNAs-induced mutations. For the mutants T0-RP1 and

T0-RP7, primers EC3-F and EC3-R were used for amplifying C3 site and the

amplicons were sequenced using EC3-F; primers EPSPS-F and EPSPS-R were used

for amplifying C5 site with the amplicons sequenced using EPSPS-F. For other

mutants, primers F1 and R1 were used and primer F2 was for sequencing. On the

right, “–” and “+” indicate the number of nucleotides deleted and inserted at the C3

and C5 sites, respectively; “ * ” indicates the numbers of independent clones




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sequenced. (d) The two sgRNAs generating gene deletions, inversions and insertions

via the NHEJ pathway. (e) PCR analysis of T0-RP1 and T0-RP7 using different

primers. Primer Ein-F is in the deletion region (shown in d). PCR reactions were

performed using three different combinations of primers, i.e., F1/R1, F1/Ein-F/R1 or

Ein-F/R1. The primer pair of F1 and R1 can amplify both wt allele (big band) and

deletion allele. The other primer pair of Ein-F and R1 can only amplify the wt allele

(small band) in T0-RP1 and T0-RP7.




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ggtactaaatatacaatcccttgggtttatttgtttccaagcatgtcattaacttatcttaatgtggacaagaaactgatgcctgcttacattgctattatttcaagcgggtattgatcctttgacatgtgattgatcatttttttttctctggttattagGGCACAACAGTGGTGGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAGCCCTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCTGTGGTGGCAAGTTTCCTGTTGAGAAGGATGCGAAAGAGGAAGTGCAACTCTTCTTGGGGAACGCTGGAATTGCAATGCGATCGTTGACAGCAGCCGTGACTGCTGCTGGTGGAAATGCAACTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTGGTGACTTGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGCACTGAATGCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGGTTAAGCTCTCTGGTTCCATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCTCCTTTGGCCCTTGGGGATGTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCTTACGTTGAAATGACATTGAGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTCTGATAGTTGGGACAGATTCTATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAAATGCCTATGTTGAAGGTGATGCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCAATCACTGGAGGCACTGTGACAGTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTGATGTCAAATTTGCTGAGGTACTTGAGATGATGGGAGCAAAGGTTACATGGACTGACACCAGTGTAACCGTAACTGGTCCACCACGTGAGCCTTATGGGAAGAAACACCTGAAAGCTGTTGATGTCAACATGAACAAAATGCCTGATGTTGCCATGACCCTTGCCGTTGTTGCACTCTTCGCTGATGGTCCAACTGCTATCAGAGATGTGGCTTCCTGGAGAGTAAAGGAAACCGAAAGGATGGTTGCAATTCGGACCGAGCTAACAAAGCTGGGAGCATCGGTTGAAGAAGGTCCTGACTACTGCATCATCACCCCACCGGAGAAGCTGAACATCACGGCAATCGACACCTACGATGATCACAGGATGGCCATGGCCTTCTCCCTCGCTGCCTGCGCCGACGTGCCCGTGACGATCAGGGACCCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCACTTTCGTCAGGAACTGAACTGAGCTTTTAAAAGAGTGAGGTCTAGGTTCTGTTGTCTGTCTGTCCATCATCCATGTGTTGACTGTTGAGGGAACTCGTTTCTTCTTTTCTTCACGAGATGAGTTTTTGTGTGCCTGTAATACTAGTTTGTAGCAAAGGCTGCGTTACATAAGGTGATGAGAATTGAGGTAAAATGAGATCTGTACACTAAATTCATTCAGACTGTTTTGGCATAAAGAATAATTTGGCCTTCTGCGATTTCAGAAGCTATAAATTGCCATCTCACTAAAtactaaatatacaatcccttggg

Supplementary Figure 5. DNA sequence of the E2-TIPS-E8 donor template. Exons

and 3’-UTR are in upper case letters and intron is in lower case letters. The sgRNA

C3 site is marked in green. The DNA sequence of the conserved motif is underlined

and the 3 nucleotide substitutions are highlighted in red.




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Supplementary Figure 6. Outcome of PCR/RE assays to detect C3 sgRNA-induced

mutations in 14 T0 mutant plants. Lanes T0-IP1 to T0-IP14, PCR fragments amplified

from the transgenic rice plants digested with BsaJI. The primers used for C3 site

amplification are EC3-F and EC3-R. Lanes wt, PCR fragments amplified from a wild

type control plant with and without BsaJI digestion. The bands marked by red

arrowheads are caused by the sgRNA-induced mutations. The sequences of the

sgRNA-induced mutations in the transgenic T0-RP1 to T0-RP14 plants are shown.

The wild-type sequence is shown at the top. The sgRNA target sequence (C3) is

underlined and the PAM is in green. On the right, “–” and “+” indicate the number of

nucleotides deleted and inserted at the C3 site; “ * ” indicates the numbers of

independent clones sequenced.




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ATGGCGGCGACCATGGCGTCCAACGCCGCGGCTGCGGCGGCGGTGTCCCTGGACCAGGCCGTGGCGGCGTCGGCGGCGTTCTCGTCGCGGAAGCAGCTGCGGCTGCCCGCCGCGGCGCGCGGGGGGATGCGGGTGCGGGTGCGGGCGCGGGGGCGGCGGGAGGCGGTGGTGGTGGCGTCCGCGTCGTCGTCGTCGGTGGCAGCGCCGGCGGCGAAGGCGGAGGAGATCGTGCTCCAGCCCATCAGGGAGATCTCCGGGGCGGTTCAGCTGCCAGGGTCCAAGTCGCTCTCCAACAGGATCCTCCTCCTCTCCGCCCTCTCCGAGGGCACAACAGTGGTGGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAGCCCTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCTGTGGTGGCAAGTTTCCTGTTGAGAAGGATGCGAAAGAGGAAGTGCAACTCTTCTTGGGGAACGCTGGAATTGCAATGCGATCGTTGACAGCAGCCGTGACTGCTGCTGGTGGAAATGCAACTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTGGTGACTTGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGCACTGAATGCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGGTTAAGCTCTCTGGTTCCATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCTCCTTTGGCCCTTGGGGATGTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCTTACGTTGAAATGACATTGAGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTCTGATAGTTGGGACAGATTCTATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAAATGCCTATGTTGAAGGTGATGCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCAATCACTGGAGGCACTGTGACAGTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTGATGTCAAATTTGCTGAGGTACTTGAGATGATGGGAGCAAAGGTTACATGGACTGACACCAGTGTAACCGTAACTGGTCCACCACGTGAGCCTTATGGGAAGAAACACCTGAAAGCTGTTGATGTCAACATGAACAAAATGCCTGATGTTGCCATGACCCTTGCCGTTGTTGCACTCTTCGCTGATGGTCCAACTGCTATCAGAGATGTGGCTTCCTGGAGAGTAAAGGAAACCGAAAGGATGGTTGCAATTCGGACCGAGCTAACAAAGCTGGGAGCATCGGTTGAAGAAGGTCCTGACTACTGCATCATCACCCCACCGGAGAAGCTGAACATCACGGCAATCGACACCTACGATGATCACAGGATGGCCATGGCCTTCTCCCTCGCTGCCTGCGCCGACGTGCCCGTGACGATCAGGGACCCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCACTTTCGTCAGGAACTGA

Supplementary Figure 7. Sequences of the ORFs of EPSPS from the TIPS mutants.

Sequence result with the nucleotides substitutions from the TIPS mutants obtained by

gene replacement (RP) and gene insertion (IP). It has eight exons. The initiation

codon (ATG) and termination codon (TGA) are underlined. Exon 2 contains the 3

nucleotide substitutions (C518T, C529T and A531G), highlighted in red.




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Supplementary Figure 8. Genotypes of the T1 progeny from the T0 plants with point mutations and exon 2 deletion. (a) PCR/RE assay of the T1

progeny from T0-RP4, T0-IP8 and T0-RP1. The bands marked by red arrowheads indicate TIPS substitutions. The bands marked by green

horizontal arrows indicate exon 2 deletions. (b) The sequencing result of the T1 progenies from T0-RP4, T0-IP8 and T0-RP1. Five T1 mutant

plants from each T0 line were sequenced and the results were the same as their T0 plant mutations. The sizes of the indels are given to the right of

each sequence. The 3 nucleotide substitutions (C518-T, C529-T and A531-G) are highlighted in red.




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Supplementary Figure 8. Genotypes of the T1 progeny from the T0 plants with point mutations and exon 2 deletion. (a) PCR/RE assay of the T1

progeny from T0-RP4, T0-IP8 and T0-RP1. The bands marked by red arrowheads indicate TIPS substitutions. The bands marked by green

horizontal arrows indicate exon 2 deletions. (b) The sequencing result of the T1 progenies from T0-RP4, T0-IP8 and T0-RP1. Five T1 mutant

plants from each T0 line were sequenced and the results were the same as their T0 plant mutations. The sizes of the indels are given to the right of

each sequence. The 3 nucleotide substitutions (C518-T, C529-T and A531-G) are highlighted in red.

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Supplementary Figure 9. The seed-setting rates of the T0 mutant plants. wt, wild

type; plant T0-RP3, with homozygous mutations at both C3 and C5; plant T0-IP1,

with homozygous mutations at C3; plant T0-RP4, with TIPS mutations; plant T0-RP6,

with TIPS mutations; plant T0-RP8, with TIPS mutations; plant T0-RP10, with TIPS

mutations; plant T0-IP8, with TIPS mutations obtained by targeted insertion; plant

T0-IP11, with TIPS mutations obtained by targeted insertion; plant T0-RP1, with

exon 2 deletion; plant T0-RP7, with exon 2 deletion.




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Supplementary Figure 10. Intron-mediated gene replacement with GFP coding

sequence at OsDEP1 locus via NHEJ pathway in rice protoplasts. (a) Scheme of gene

replacement with GFP coding sequence at OsDEP1 locus. The sgRNA sites (D1 and

D2), targeting the promoter region and intron 1 of OsDEP1, respectively, are

indicated in orange or purple. The targeting vector pHUN411-D1D2 and the donor

plasmid (TB-D1GFPD2) with GFP coding sequence flanked by the D1 and D2

sgRNA sites were co-transformed into rice protoplasts, generating GFP replacement

for the fragment between the D1 and D2 site in DEP1. DF1, DR1, DF2 and DR2 are

four primers used for mutation analysis (see below). (b) PCR analysis and sequencing

of the 5’ and 3’ junctions of the mutations associated with the replacement. Primers




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DF1 and DR1 were used for 5’ junction analysis and DF2 and DR2 for 3’ junction

analysis. The newly formed 5’ and 3’ junctions were amplified by PCR only if the

fragment between the D1 and D2 sites in OsDEP1 is replaced with GFP coding

sequence in protoplasts. The hypothetical sequences (HS) of 5’ junction and 3’

junction are indicated if the fragment between the D1 and D2 sites in OsDEP1 is

replaced with GFP coding sequence through NHEJ pathway. The PAM is in bold

typeface and the three nucleotides left of D1 or D2 are indicated by orange or purple,

respectively. The PCR products containing the 5’ or 3’ junctions were cloned and

sequenced. The coding sequence of GFP is in green capitals. On the right, “–” and “+”

indicate the number of nucleotides deleted and inserted.




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Target site ID Gene Target Site sequence Oligo-F (5’-3’) Oligo-R(5’-3’) Enzyme site Target region

C1 OsEPSPS GGCTGTGTTTGTGAAATCCTAGG GGCGGGCTGTGTTTGTGAAATCCT AAACAGGATTTCACAAACACAGCC AvrII Intron 1

C2 OsEPSPS ATGATATCCTCCTACATGTCAGG GGCGATGATATCCTCCTACATGTC AAACGACATGTAGG AGGATATCAT PciI Intron 1

C3 OsEPSPS TACTAAATATACAATCCCTTGGG GGCGTACTAAATATACAATCCCTT AAACAAGGGATTGTATATTTAGTA BsaJI Intron 1

C4 OsEPSPS GAAAATATGTATGGAATTCATGG GGCGGAAAATATGTATGGAATTCA AAACTGAATTCCATACATATTTTC EcoRI Intron 2

C5 OsEPSPS AAAATATGTATGGAATTCATGGG GGCGAAAATATGTATGGAATTCAT AAACATGAATTCCATACATATTTT EcoRI Intron 2

C6 OsEPSPS GAGGAAGTGCAACTCTTCTTGGG GGCGGAGGAAGTGCAACTCTTCTT AAACAAGAAGAGTTGCACTTCCTC EarI Exon 2

D1 OsDEP1 CCGCTAGCTCGATCCGCCTCGT GGCGACGAGGCGGATCGAGCTAG AAACCTAGCTCGATCCGCCTCGT NheI Promoter

D2 OsDEP1 GAGGCTGGCGAGACAAGCTTGG GGCGGAGGCTGGCGAGACAAGCT AAACAGCTTGTCTCGCCAGCCTC HindIII Intron 1

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Supplementary Table 2. Primers used in this study.

Primer name Primer sequence Experiment F1, EC12-R 5’-CAGCCCATCAGGGAGATCTC-3’, 5’-TTGGGAGAAAGCACTGCTTGC-3’ Amplifying the sequence containing C1 and C2

EC3-F, EC3-R 5’-GCTGTGTTTGTGAAATCCTAGG-3’, 5’- GAGGGCTTTCAGGGCCTCAAG -3’ Amplifying the sequence containing C3 and sequencing

EPSPS-F, EPSPS-R 5’-ATGTGGACAAGAAACTGATGCC-3’, 5’-TAACCTTGCCACCAGGAAGTC-3’ Amplifying the sequence containing C4 and C5and sequencing

F2, R1 5’-TATAATTCCCCAATACATTGCTC-3’, 5’-TAACCTTGCCACCAGGAAGTC-3’ Amplifying the sequence containing C6

E-TIPSmut-F

E-TIPSmut-R

5’-GAACGCTGGAATTGCAATGCGATCGTTGACAGCAGCCGTGAC-3’,

5’-TGCTGTCAACGATCGCATTGCAATTCCAGCGTTCCCCAAG-3’ Site-directed mutagenesis of exon 2

ED-1F, ED-1R 5’-CCCTCTCCGAGGTGAGACG-3’, 5’-TCCAATTCCCTTGACACGAAC-3’ Constructing the donor vector TB-TIPS-E2

ED-3F1

ED-3R1

ED-3F2

ED-3R2

5’-AGGTATGATATCCTCCTACATGTCAG-3’,

5’-ATCAAGCACATAATTGCATTTCCACCAGCAGCAG-3’

5’-TGGAAATGCAACTTATGTGCTTGATGGAGTGCC-3’,

5’-CCCATGAATTCCATACATATTTTCTTTAGTGAGATGGCAATTTATAGC-3’

Constructing the donor vector TB-E2-TIPS-E8

F1, R1 5’-CAGCCCATCAGGGAGATCTC-3’, 5’-TAACCTTGCCACCAGGAAGTC-3’ Amplifying the conserved motif and detecting the deletion

F1, R4 5’-CAGCCCATCAGGGAGATCTC-3’, 5’-CTGTTGAGTGTAATTCAGTTAAATGC-3’ Detecting the entire sequence (1.6kb) insertion

F2 5’-TATAATTCCCCAATACATTGCTC-3’ Sequencing the point mutations and C3, C5 indels

F2, R2 5’-TATAATTCCCCAATACATTGCTC-3’, 5’-TTCCAGGAGATTTGTACTTCTGC-3’ Detecting NHEJ-mediated E2-TIPS-E8 inserts

F3, R3 5’-GGTGATGAGAATTGAGGTAAAATGAG-3’, 5’-CACTGTTGTGCCCTAATAACCAG-3’ Detecting NHEJ-mediated E2-TIPS-E8 inserts

ATG-F, TGA-R 5’-ATGGCGGCGACCATGGCGTC-3’, 5’-TCAGTTCCTGACGAAAGTGCTTAG-3’ Amplifying the full cDNA of the EPSPS gene

qRT-EPSPS-F, qRT-EPSPS-R 5’-CTCTGTGGAAGCAGATAAAGTTGC-3’, 5’-CATCAAGCACATAAGTTGCATTTC-3’ Quantitative real-time PCR for EPSPS

qRT-ACTIN-F , RT-ACTIN-R 5’-GACCCAGATCATGTTTGAGACC-3’, 5’-CATCACCAGAGTCCAACACAATAC-3’ Quantitative real-time PCR for ACTIN

HPT-F, HPT-R 5’-CACTATCCTTCGCAAGACCTTC-3’, 5’-CAATCGCGCATATGAAATCAC-3’ Detecting the presence of HPT

Cas9-1F, Cas9-1R 5’-GGCCGTGATCACCGATGAGTAC-3’, 5’-TATCACTCAGGAGGATCGCGTC-3’ Detecting the presence of Cas9

C3-OT1F, C3-OT1R 5’-AGGAGTTGAATCACCTAAGGTG-3’, 5’-GGTTTAGGGTTTAGCTTGAGC-3’ Off-target detecting of the C3-OT1

C3-OT2F, C3-OT2R 5’-ACCTCAAATTGCATACCTTCTG-3’, 5’-TCTGCTACAACCAACAGCATC-3’ Off-target detecting of the C3-OT2

C3-OT3F, C3-OT3R 5’-AACTGACCGAGTTCAAGGAGG-3’, 5’-TGATAGGACGTGCAAATCCTC-3’ Off-target detecting of the C3-OT3




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Target site ID Gene Target Site sequence Oligo-F (5’-3’) Oligo-R(5’-3’) Enzyme site Target region

C1 OsEPSPS GGCTGTGTTTGTGAAATCCTAGG GGCGGGCTGTGTTTGTGAAATCCT AAACAGGATTTCACAAACACAGCC AvrII Intron 1

C2 OsEPSPS ATGATATCCTCCTACATGTCAGG GGCGATGATATCCTCCTACATGTC AAACGACATGTAGG AGGATATCAT PciI Intron 1

C3 OsEPSPS TACTAAATATACAATCCCTTGGG GGCGTACTAAATATACAATCCCTT AAACAAGGGATTGTATATTTAGTA BsaJI Intron 1

C4 OsEPSPS GAAAATATGTATGGAATTCATGG GGCGGAAAATATGTATGGAATTCA AAACTGAATTCCATACATATTTTC EcoRI Intron 2

C5 OsEPSPS AAAATATGTATGGAATTCATGGG GGCGAAAATATGTATGGAATTCAT AAACATGAATTCCATACATATTTT EcoRI Intron 2

C6 OsEPSPS GAGGAAGTGCAACTCTTCTTGGG GGCGGAGGAAGTGCAACTCTTCTT AAACAAGAAGAGTTGCACTTCCTC EarI Exon 2

D1 OsDEP1 CCGCTAGCTCGATCCGCCTCGT GGCGACGAGGCGGATCGAGCTAG AAACCTAGCTCGATCCGCCTCGT NheI Promoter

D2 OsDEP1 GAGGCTGGCGAGACAAGCTTGG GGCGGAGGCTGGCGAGACAAGCT AAACAGCTTGTCTCGCCAGCCTC HindIII Intron 1

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Supplementary Table 2. Primers used in this study.

Primer name Primer sequence Experiment F1, EC12-R 5’-CAGCCCATCAGGGAGATCTC-3’, 5’-TTGGGAGAAAGCACTGCTTGC-3’ Amplifying the sequence containing C1 and C2

EC3-F, EC3-R 5’-GCTGTGTTTGTGAAATCCTAGG-3’, 5’- GAGGGCTTTCAGGGCCTCAAG -3’ Amplifying the sequence containing C3 and sequencing

EPSPS-F, EPSPS-R 5’-ATGTGGACAAGAAACTGATGCC-3’, 5’-TAACCTTGCCACCAGGAAGTC-3’ Amplifying the sequence containing C4 and C5and sequencing

F2, R1 5’-TATAATTCCCCAATACATTGCTC-3’, 5’-TAACCTTGCCACCAGGAAGTC-3’ Amplifying the sequence containing C6

E-TIPSmut-F

E-TIPSmut-R

5’-GAACGCTGGAATTGCAATGCGATCGTTGACAGCAGCCGTGAC-3’,

5’-TGCTGTCAACGATCGCATTGCAATTCCAGCGTTCCCCAAG-3’ Site-directed mutagenesis of exon 2

ED-1F, ED-1R 5’-CCCTCTCCGAGGTGAGACG-3’, 5’-TCCAATTCCCTTGACACGAAC-3’ Constructing the donor vector TB-TIPS-E2

ED-3F1

ED-3R1

ED-3F2

ED-3R2

5’-AGGTATGATATCCTCCTACATGTCAG-3’,

5’-ATCAAGCACATAATTGCATTTCCACCAGCAGCAG-3’

5’-TGGAAATGCAACTTATGTGCTTGATGGAGTGCC-3’,

5’-CCCATGAATTCCATACATATTTTCTTTAGTGAGATGGCAATTTATAGC-3’

Constructing the donor vector TB-E2-TIPS-E8

F1, R1 5’-CAGCCCATCAGGGAGATCTC-3’, 5’-TAACCTTGCCACCAGGAAGTC-3’ Amplifying the conserved motif and detecting the deletion

F1, R4 5’-CAGCCCATCAGGGAGATCTC-3’, 5’-CTGTTGAGTGTAATTCAGTTAAATGC-3’ Detecting the entire sequence (1.6kb) insertion

F2 5’-TATAATTCCCCAATACATTGCTC-3’ Sequencing the point mutations and C3, C5 indels

F2, R2 5’-TATAATTCCCCAATACATTGCTC-3’, 5’-TTCCAGGAGATTTGTACTTCTGC-3’ Detecting NHEJ-mediated E2-TIPS-E8 inserts

F3, R3 5’-GGTGATGAGAATTGAGGTAAAATGAG-3’, 5’-CACTGTTGTGCCCTAATAACCAG-3’ Detecting NHEJ-mediated E2-TIPS-E8 inserts

ATG-F, TGA-R 5’-ATGGCGGCGACCATGGCGTC-3’, 5’-TCAGTTCCTGACGAAAGTGCTTAG-3’ Amplifying the full cDNA of the EPSPS gene

qRT-EPSPS-F, qRT-EPSPS-R 5’-CTCTGTGGAAGCAGATAAAGTTGC-3’, 5’-CATCAAGCACATAAGTTGCATTTC-3’ Quantitative real-time PCR for EPSPS

qRT-ACTIN-F , RT-ACTIN-R 5’-GACCCAGATCATGTTTGAGACC-3’, 5’-CATCACCAGAGTCCAACACAATAC-3’ Quantitative real-time PCR for ACTIN

HPT-F, HPT-R 5’-CACTATCCTTCGCAAGACCTTC-3’, 5’-CAATCGCGCATATGAAATCAC-3’ Detecting the presence of HPT

Cas9-1F, Cas9-1R 5’-GGCCGTGATCACCGATGAGTAC-3’, 5’-TATCACTCAGGAGGATCGCGTC-3’ Detecting the presence of Cas9

C3-OT1F, C3-OT1R 5’-AGGAGTTGAATCACCTAAGGTG-3’, 5’-GGTTTAGGGTTTAGCTTGAGC-3’ Off-target detecting of the C3-OT1

C3-OT2F, C3-OT2R 5’-ACCTCAAATTGCATACCTTCTG-3’, 5’-TCTGCTACAACCAACAGCATC-3’ Off-target detecting of the C3-OT2

C3-OT3F, C3-OT3R 5’-AACTGACCGAGTTCAAGGAGG-3’, 5’-TGATAGGACGTGCAAATCCTC-3’ Off-target detecting of the C3-OT3




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Supplementary Table 2. Primers used in this study. (Continued)

Primer name Primer sequence Experiment C3-OT4F, C3-OT4R 5’-AGGCTATGGTCGAGGCTACTG-3’, 5’-CATAGGGGAAGTGCTACATCG-3’ Off-target detecting of the C3-OT4

C3-OT5F, C3-OT5R 5’-CATTGTTTGTGGAGAGGATCTG-3’, 5’-GCCTGTTCTGACAAGGTAAGTC-3’ Off-target detecting of the C3-OT5

C3-OT6F, C3-OT6R 5’-GCCTTTCATCCAATATCATCAC-3’, 5’-GTTACAGGTCCTGTTACCATCG-3’ Off-target detecting of the C3-OT6

C5-OT1F, C5-OT1R 5’-CACCGACTTCTTTGATTCTCC-3’, 5’-GGTGTCCACTAAAACTTGAATTG-3’ Off-target detecting of the C5-OT1

C5-OT2F, C5-OT2R 5’-TATACACACATCCGCAAGTGG-3’, 5’-GAAGCGTGTTCACTGAATATGG-3’ Off-target detecting of the C5-OT2

C5-OT3F, C5-OT3R 5’-GTCCACCGGACCGCCTTTATC-3’, 5’-GAGGCCAAGCAAGTTATATGTGG-3’ Off-target detecting of the C5-OT3

C5-OT4F, C5-OT4R 5’-CTGAATCTAACCCATGGGTTC-3’, 5’-GTCCACTTGGTGAGGTAGACTC-3’ Off-target detecting of the C5-OT4

C5-OT5F, C5-OT5R 5’-GGAATTTGCTTGCGTTCGTTC-3’, 5’-AGCAACGCTAAGAGAGCACCAC-3’ Off-target detecting of the C5-OT5

DF1, DR1 5’-CTCTCTCCAAACCCCACGCAC-3’, 5’-TTGTGGCGGATCTTGAAGTTC-3’ Detecting NHEJ-mediated gene replacement in OsDEP1

DF2, DR2 5’-GGGCACAAGCTGGAGTACAAC-3’, 5’-GCTATGGCTCTCCCTGACAAG-3’ Detecting NHEJ-mediated gene replacement in OsDEP1

F1, Ein-F, R1 5’-CAGCCCATCAGGGAGATCTC-3’, 5’-ATGTGGACAAGAAACTGATGC-3’,

5’-TAACCTTGCCACCAGGAAGTC-3’ Detecting the deletion mutations




16 / 20

Supplementary Table 2. Primers used in this study. (Continued)

Primer name Primer sequence Experiment C3-OT4F, C3-OT4R 5’-AGGCTATGGTCGAGGCTACTG-3’, 5’-CATAGGGGAAGTGCTACATCG-3’ Off-target detecting of the C3-OT4

C3-OT5F, C3-OT5R 5’-CATTGTTTGTGGAGAGGATCTG-3’, 5’-GCCTGTTCTGACAAGGTAAGTC-3’ Off-target detecting of the C3-OT5

C3-OT6F, C3-OT6R 5’-GCCTTTCATCCAATATCATCAC-3’, 5’-GTTACAGGTCCTGTTACCATCG-3’ Off-target detecting of the C3-OT6

C5-OT1F, C5-OT1R 5’-CACCGACTTCTTTGATTCTCC-3’, 5’-GGTGTCCACTAAAACTTGAATTG-3’ Off-target detecting of the C5-OT1

C5-OT2F, C5-OT2R 5’-TATACACACATCCGCAAGTGG-3’, 5’-GAAGCGTGTTCACTGAATATGG-3’ Off-target detecting of the C5-OT2

C5-OT3F, C5-OT3R 5’-GTCCACCGGACCGCCTTTATC-3’, 5’-GAGGCCAAGCAAGTTATATGTGG-3’ Off-target detecting of the C5-OT3

C5-OT4F, C5-OT4R 5’-CTGAATCTAACCCATGGGTTC-3’, 5’-GTCCACTTGGTGAGGTAGACTC-3’ Off-target detecting of the C5-OT4

C5-OT5F, C5-OT5R 5’-GGAATTTGCTTGCGTTCGTTC-3’, 5’-AGCAACGCTAAGAGAGCACCAC-3’ Off-target detecting of the C5-OT5

DF1, DR1 5’-CTCTCTCCAAACCCCACGCAC-3’, 5’-TTGTGGCGGATCTTGAAGTTC-3’ Detecting NHEJ-mediated gene replacement in OsDEP1

DF2, DR2 5’-GGGCACAAGCTGGAGTACAAC-3’, 5’-GCTATGGCTCTCCCTGACAAG-3’ Detecting NHEJ-mediated gene replacement in OsDEP1

F1, Ein-F, R1 5’-CAGCCCATCAGGGAGATCTC-3’, 5’-ATGTGGACAAGAAACTGATGC-3’,

5’-TAACCTTGCCACCAGGAAGTC-3’ Detecting the deletion mutations

17 / 20

Supplementary Table 3. Potential off-target sites of C3 and C5 detected in rice

protoplasts and TIPS T0 plants.

ID Target and off-target sequence No. of

mismatches

Mutations detected in protoplasts

and TIPS plants

C3 site TACTAAATATACAATCCCTTGGG on target +

C3-OT1 TttTtAATATAaAATCCCTTGGG 4 -

C3-OT2 TAtTAgATATAaAAaCCCTTGGG 4 -

C3-OT3 TAaTAAAcAgACAATtCCTTGGG 4 -

C3-OT4 aAtTAAAaATACAATaCCTTGGG 4 -

C3-OT5 TAaTAgAcATcCtATCCCTTGGG 5 -

C3-OT6 TcgTAAAgATAttATCCCTTGGG 5 -

C5 site AAAATATGTATGGAATTCATGGG on target +

C5-OT1 gAAATATaTATtGAATTCATGGG 3 -

C5-OT2 cAAATtTGTAaGGAATTCATTGG 3 -

C5-OT3 AAAtgATtTATGGAATTCATGGG 3 -

C5-OT4 ggAgTATGTAaGGAATTCATTGG 4 -

C5-OT5 AAAATActTtgGGAATTCATGGG 4 -

Red lowercase bases are mismatches to C3 or C5; +, mutations detected; -, mutations are not

detected.




18 / 20

Supplementary Table 4. Genetic analysis of T0 plants with homozygous mutation at C3 and C5 or C3 and their transmissions to the T1

generation.

Plant ID Genotype

(C3, C5 or C3 (bp) ) No. of tested plants

Genetic segregation in T1 population Homozygous Genotype

(C3, C5 or C3 (bp) ) Wild type Heterozygote Homozygote

T0-RP3 C3 (+1), C5 (-2) 96 0 0 96 C3 (+1), C5 (-2)

T0-IP1 C3 (+1) 96 0 0 96 C3 (+1)

19 / 20

Supplementary Table 5. Genetic analysis of intron targeting-mediated T0 mutations in OsEPSPS and their transmissions to the T1 generation.

Plant ID Genotypes at

C3, Exon 2, C5 (bp) No. of tested

plants

Genetic segregation in T1 population for TIPS point mutations * Wild type Heterozygote Homozygote

No. Genotypes at

C3, Exon 2, C5 (bp) No.

Genotypes at C3, Exon 2, C5 (bp)

No.

T0-RP4 +1, Exon 2, +1

+1, TIPS, -20 96 41

+1, Exon 2, +1

+1, Exon 2, +1 55

+1, Exon 2, +1

+1, TIPS, -20 0

T0-RP6 +1, Exon 2, -2

+1, TIPS, -4 96 38

+1, Exon 2, -2

+1, Exon 2, -2 58

+1, Exon 2, -2

+1, TIPS, -4 0

T0-RP8 +1, Exon 2, -2

+1, TIPS, -4 96 36

+1, Exon 2, -2

+1, Exon 2, -2 60

+1, Exon 2, -2

+1, TIPS, -4 0

T0-RP10 +1, Exon 2, +1

+1, TIPS, -3 96 82

+1, Exon 2, +1

+1, Exon 2, +1 14

+1, Exon 2, +1

+1, TIPS, -3 0

T0-IP8 -19/+1, Exon 2, ---

+77, IN,--- 96 40

-19/+1, Exon 2, ---

-19/+1, Exon 2, --- 56

-19/+1, Exon 2, ---

+77, IN,--- 0

T0-IP11 -22, Exon 2, ---

-28, IN, --- 96 34

-22, Exon 2, ---

-22, Exon 2, --- 62

-22, Exon 2, ---

-28, IN, --- 0

T0-RP1 +1, Exon 2, +1

-429 96 79

+1, Exon 2, +1

+1, Exon 2, +1 17

+1, Exon 2, +1

-429 0

T0-RP7 +2, Exon 2, -19

-435 96 57

+2, Exon 2, -19

+2, Exon 2, -19 39

+2, Exon 2, -19

-435 0

*, all those mutations carrying homozygous mutations at both C3 and C5 or at C3; IN, insertion; -n, deletion of the indicated number of nucleotides; +n, insertion of

indicated number of nucleotides; -n/+n, simultaneous deletion and insertion of the indicated number of nucleotides at the same site. --- indicating not applicable.




18 / 20

Supplementary Table 4. Genetic analysis of T0 plants with homozygous mutation at C3 and C5 or C3 and their transmissions to the T1

generation.

Plant ID Genotype

(C3, C5 or C3 (bp) ) No. of tested plants

Genetic segregation in T1 population Homozygous Genotype

(C3, C5 or C3 (bp) ) Wild type Heterozygote Homozygote

T0-RP3 C3 (+1), C5 (-2) 96 0 0 96 C3 (+1), C5 (-2)

T0-IP1 C3 (+1) 96 0 0 96 C3 (+1)

19 / 20

Supplementary Table 5. Genetic analysis of intron targeting-mediated T0 mutations in OsEPSPS and their transmissions to the T1 generation.

Plant ID Genotypes at

C3, Exon 2, C5 (bp) No. of tested

plants

Genetic segregation in T1 population for TIPS point mutations * Wild type Heterozygote Homozygote

No. Genotypes at

C3, Exon 2, C5 (bp) No.

Genotypes at C3, Exon 2, C5 (bp)

No.

T0-RP4 +1, Exon 2, +1

+1, TIPS, -20 96 41

+1, Exon 2, +1

+1, Exon 2, +1 55

+1, Exon 2, +1

+1, TIPS, -20 0

T0-RP6 +1, Exon 2, -2

+1, TIPS, -4 96 38

+1, Exon 2, -2

+1, Exon 2, -2 58

+1, Exon 2, -2

+1, TIPS, -4 0

T0-RP8 +1, Exon 2, -2

+1, TIPS, -4 96 36

+1, Exon 2, -2

+1, Exon 2, -2 60

+1, Exon 2, -2

+1, TIPS, -4 0

T0-RP10 +1, Exon 2, +1

+1, TIPS, -3 96 82

+1, Exon 2, +1

+1, Exon 2, +1 14

+1, Exon 2, +1

+1, TIPS, -3 0

T0-IP8 -19/+1, Exon 2, ---

+77, IN,--- 96 40

-19/+1, Exon 2, ---

-19/+1, Exon 2, --- 56

-19/+1, Exon 2, ---

+77, IN,--- 0

T0-IP11 -22, Exon 2, ---

-28, IN, --- 96 34

-22, Exon 2, ---

-22, Exon 2, --- 62

-22, Exon 2, ---

-28, IN, --- 0

T0-RP1 +1, Exon 2, +1

-429 96 79

+1, Exon 2, +1

+1, Exon 2, +1 17

+1, Exon 2, +1

-429 0

T0-RP7 +2, Exon 2, -19

-435 96 57

+2, Exon 2, -19

+2, Exon 2, -19 39

+2, Exon 2, -19

-435 0

*, all those mutations carrying homozygous mutations at both C3 and C5 or at C3; IN, insertion; -n, deletion of the indicated number of nucleotides; +n, insertion of

indicated number of nucleotides; -n/+n, simultaneous deletion and insertion of the indicated number of nucleotides at the same site. --- indicating not applicable.




20 / 20

Supplementary Table 6. Analysis of the gene replacement, targeted gene insertion and Cas9-free in the T1 generation.

Plant ID No. of tested plants Genetic segregation in T1 population for TIPS point mutations

Wild type Heterozygote Homozygote Cas9-free* (%)

T0-RP4 81 34 47 0 3/47 (6.4)

T0-RP6 84 33 51 0 7/51 (13.7)

T0-RP8 63 25 38 0 2/38 (5.3)

T0-RP10 45 36 9 0 0/9 (0)

T0-IP8 48 20 28 0 2/28 (7.1)

T0-IP11 38 15 23 0 1/23 (4.3)

*, absence of Cas9 and hpt genes, based on the number of the gene replacement/targeted gene insertion plants harboring neither the Cas9 nor hpt gene over the total

number of the gene replacement/targeted gene insertion plants tested.


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SUPPLEMENTARY INFORMATION - Nature Research...DOI: 10.1038/NPLANTS.2016.139 SUPPLEENTAR INORATION 10 / 20 exon 2 deletion Supplementary Figure 8. Genotypes of the T 1 progeny from