SUPPLEMENTARY INFORMATION

Transcription

1 Gene replacements and insertions in rice by intron targeting using CRISPR Cas9 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 sgrnainduced mutations in 14 T 0 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 T 0 mutant plants. Supplementary Figure 7. Sequences of the ORFs of EPSPS from the TIPS mutants. Supplementary Figure 8. Genotypes of the T 1 progeny from the T 0 plants with point mutations and exon 2 deletion. Supplementary Figure 9. The seed-setting rates of the T 0 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 T 0 plants. Supplementary Table 4. Genetic analysis of T 0 plants with homozygous mutation at C3 and C5 or C3 and their transmissions to the T 1 generation. Supplementary Table 5. Genetic analysis of intron targeting-mediated T 0 mutations in OsEPSPS and their transmissions to the T 1 generation. Supplementary Table 6. Analysis of the gene replacement, targeted gene insertion and Cas9-free in the T 1 generation. NATURE PLANTS 1

2 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. 2 NATURE PLANTS

3 SUPPLEMENTARY INFORMATION 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. NATURE PLANTS 3

4 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. 4 NATURE PLANTS

5 SUPPLEMENTARY INFORMATION Supplementary Figure 4. Outcome of PCR/RE assays to detect C3 and C5 sgrnainduced 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 NATURE PLANTS 5

6 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. 6 NATURE PLANTS

7 SUPPLEMENTARY INFORMATION ggtactaaatatacaatcccttgggtttatttgtttccaagcatgtcattaacttat cttaatgtggacaagaaactgatgcctgcttacattgctattatttcaagcgggtat tgatcctttgacatgtgattgatcatttttttttctctggttattagggcacaacag TGGTGGACAACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAG CCCTCGGGCTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCT GTGGTGGCAAGTTTCCTGTTGAGAAGGATGCGAAAGAGGAAGTGCAACTCTTCTTGG GGAACGCTGGAATTGCAATGCGATCGTTGACAGCAGCCGTGACTGCTGCTGGTGGAA ATGCAACTTATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTGGTGACT TGGTTGTCGGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGCACTGAAT GCCCACCTGTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGGTTAAGCTCT CTGGTTCCATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCTCCTTTGGCCC TTGGGGATGTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCTTACGTTGAAA TGACATTGAGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTCTGATAGTTGGG ACAGATTCTATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAAATGCCTATGTTG AAGGTGATGCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCAATCACTGGAGGCA CTGTGACAGTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTGATGTCAAATTTGCTG AGGTACTTGAGATGATGGGAGCAAAGGTTACATGGACTGACACCAGTGTAACCGTAA CTGGTCCACCACGTGAGCCTTATGGGAAGAAACACCTGAAAGCTGTTGATGTCAACA TGAACAAAATGCCTGATGTTGCCATGACCCTTGCCGTTGTTGCACTCTTCGCTGATG GTCCAACTGCTATCAGAGATGTGGCTTCCTGGAGAGTAAAGGAAACCGAAAGGATGG TTGCAATTCGGACCGAGCTAACAAAGCTGGGAGCATCGGTTGAAGAAGGTCCTGACT ACTGCATCATCACCCCACCGGAGAAGCTGAACATCACGGCAATCGACACCTACGATG ATCACAGGATGGCCATGGCCTTCTCCCTCGCTGCCTGCGCCGACGTGCCCGTGACGA TCAGGGACCCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCA CTTTCGTCAGGAACTGAACTGAGCTTTTAAAAGAGTGAGGTCTAGGTTCTGTTGTCT GTCTGTCCATCATCCATGTGTTGACTGTTGAGGGAACTCGTTTCTTCTTTTCTTCAC GAGATGAGTTTTTGTGTGCCTGTAATACTAGTTTGTAGCAAAGGCTGCGTTACATAA GGTGATGAGAATTGAGGTAAAATGAGATCTGTACACTAAATTCATTCAGACTGTTTT GGCATAAAGAATAATTTGGCCTTCTGCGATTTCAGAAGCTATAAATTGCCATCTCAC TAAAtactaaatatacaatcccttggg 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. NATURE PLANTS 7

8 Supplementary Figure 6. Outcome of PCR/RE assays to detect C3 sgrna-induced mutations in 14 T 0 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. 8 NATURE PLANTS

9 SUPPLEMENTARY INFORMATION ATGGCGGCGACCATGGCGTCCAACGCCGCGGCTGCGGCGGCGGTGTCCCTGGACCAG GCCGTGGCGGCGTCGGCGGCGTTCTCGTCGCGGAAGCAGCTGCGGCTGCCCGCCGCG GCGCGCGGGGGGATGCGGGTGCGGGTGCGGGCGCGGGGGCGGCGGGAGGCGGTGGTG GTGGCGTCCGCGTCGTCGTCGTCGGTGGCAGCGCCGGCGGCGAAGGCGGAGGAGATC GTGCTCCAGCCCATCAGGGAGATCTCCGGGGCGGTTCAGCTGCCAGGGTCCAAGTCG CTCTCCAACAGGATCCTCCTCCTCTCCGCCCTCTCCGAGGGCACAACAGTGGTGGAC AACTTGCTGAACAGTGAGGATGTTCACTACATGCTTGAGGCCCTGAAAGCCCTCGGG CTCTCTGTGGAAGCAGATAAAGTTGCAAAAAGAGCTGTAGTCGTTGGCTGTGGTGGC AAGTTTCCTGTTGAGAAGGATGCGAAAGAGGAAGTGCAACTCTTCTTGGGGAACGCT GGAATTGCAATGCGATCGTTGACAGCAGCCGTGACTGCTGCTGGTGGAAATGCAACT TATGTGCTTGATGGAGTGCCACGAATGAGGGAGAGACCGATTGGTGACTTGGTTGTC GGGTTGAAACAACTTGGTGCGGATGTCGACTGTTTCCTTGGCACTGAATGCCCACCT GTTCGTGTCAAGGGAATTGGAGGACTTCCTGGTGGCAAGGTTAAGCTCTCTGGTTCC ATCAGCAGTCAGTACTTGAGTGCCTTGCTGATGGCTGCTCCTTTGGCCCTTGGGGAT GTGGAGATCGAAATCATTGACAAACTAATCTCCATTCCTTACGTTGAAATGACATTG AGATTGATGGAGCGTTTTGGTGTGAAGGCAGAGCATTCTGATAGTTGGGACAGATTC TATATTAAGGGAGGGCAGAAGTACAAATCTCCTGGAAATGCCTATGTTGAAGGTGAT GCCTCAAGCGCGAGCTATTTCTTGGCTGGTGCTGCAATCACTGGAGGCACTGTGACA GTTCAAGGTTGTGGTACGACCAGTTTGCAGGGTGATGTCAAATTTGCTGAGGTACTT GAGATGATGGGAGCAAAGGTTACATGGACTGACACCAGTGTAACCGTAACTGGTCCA CCACGTGAGCCTTATGGGAAGAAACACCTGAAAGCTGTTGATGTCAACATGAACAAA ATGCCTGATGTTGCCATGACCCTTGCCGTTGTTGCACTCTTCGCTGATGGTCCAACT GCTATCAGAGATGTGGCTTCCTGGAGAGTAAAGGAAACCGAAAGGATGGTTGCAATT CGGACCGAGCTAACAAAGCTGGGAGCATCGGTTGAAGAAGGTCCTGACTACTGCATC ATCACCCCACCGGAGAAGCTGAACATCACGGCAATCGACACCTACGATGATCACAGG ATGGCCATGGCCTTCTCCCTCGCTGCCTGCGCCGACGTGCCCGTGACGATCAGGGAC CCTGGTTGCACCCGCAAGACCTTCCCCAACTACTTCGACGTTCTAAGCACTTTCGTC AGGAACTGA 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. NATURE PLANTS 9

10 Supplementary Figure 8. Genotypes of the T 1 progeny from the T 0 plants with point mutations and exon 2 deletion. (a) PCR/RE assay of the T 1 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 T 1 progenies from T0-RP4, T0-IP8 and T0-RP1. Five T 1 mutant plants from each T 0 line were sequenced and the results were the same as their T 0 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. 10 NATURE PLANTS

11 SUPPLEMENTARY INFORMATION Supplementary Figure 9. The seed-setting rates of the T 0 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. NATURE PLANTS 11

12 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 12 NATURE PLANTS

13 SUPPLEMENTARY INFORMATION 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. NATURE PLANTS 13

14 Supplementary Table 1. CRISPR/Cas9 target loci and sequences. 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 14 NATURE PLANTS

15 SUPPLEMENTARY INFORMATION 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 5 -GAACGCTGGAATTGCAATGCGATCGTTGACAGCAGCCGTGAC-3, Site-directed mutagenesis of exon 2 E-TIPSmut-R 5 -TGCTGTCAACGATCGCATTGCAATTCCAGCGTTCCCCAAG-3 ED-1F, ED-1R 5 -CCCTCTCCGAGGTGAGACG-3, 5 -TCCAATTCCCTTGACACGAAC-3 Constructing the donor vector TB-TIPS-E2 ED-3F1 ED-3R1 5 -AGGTATGATATCCTCCTACATGTCAG-3, 5 -ATCAAGCACATAATTGCATTTCCACCAGCAGCAG-3 Constructing the donor vector TB-E2-TIPS-E8 ED-3F2 ED-3R2 5 -TGGAAATGCAACTTATGTGCTTGATGGAGTGCC-3, 5 -CCCATGAATTCCATACATATTTTCTTTAGTGAGATGGCAATTTATAGC-3 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 NATURE PLANTS 15

16 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, Detecting the deletion mutations 5 -TAACCTTGCCACCAGGAAGTC-3 16 NATURE PLANTS

17 SUPPLEMENTARY INFORMATION Supplementary Table 3. Potential off-target sites of C3 and C5 detected in rice protoplasts and TIPS T 0 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. NATURE PLANTS 17

18 Supplementary Table 4. Genetic analysis of T 0 plants with homozygous mutation at C3 and C5 or C3 and their transmissions to the T 1 generation. Plant ID Genotype (C3, C5 or C3 (bp) ) No. of tested plants Genetic segregation in T 1 population Wild type Heterozygote Homozygote Homozygous Genotype (C3, C5 or C3 (bp) ) T0-RP3 C3 (+1), C5 (-2) C3 (+1), C5 (-2) T0-IP1 C3 (+1) C3 (+1) 18 NATURE PLANTS

19 SUPPLEMENTARY INFORMATION Supplementary Table 5. Genetic analysis of intron targeting-mediated T 0 mutations in OsEPSPS and their transmissions to the T 1 generation. Genetic segregation in T 1 population for TIPS point mutations * Plant ID Genotypes at C3, Exon 2, C5 (bp) No. of tested plants No. Wild type Heterozygote Homozygote Genotypes at Genotypes at No. No. C3, Exon 2, C5 (bp) C3, Exon 2, C5 (bp) T0-RP4 +1, TIPS, , TIPS, T0-RP6 +1, Exon 2, -2 +1, TIPS, , Exon 2, -2 +1, Exon 2, , Exon 2, -2 +1, TIPS, -4 0 T0-RP8 +1, Exon 2, -2 +1, TIPS, , Exon 2, -2 +1, Exon 2, , Exon 2, -2 +1, TIPS, -4 0 T0-RP10 +1, TIPS, , TIPS, -3 0 T0-IP8-19/+1, Exon 2, , IN, /+1, Exon 2, /+1, Exon 2, /+1, Exon 2, , IN,--- 0 T0-IP11-22, Exon 2, , IN, , Exon 2, , Exon 2, , Exon 2, , IN, T0-RP T0-RP7 +2, Exon 2, , Exon 2, , Exon 2, , Exon 2, *, 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. NATURE PLANTS 19

20 Supplementary Table 6. Analysis of the gene replacement, targeted gene insertion and Cas9-free in the T 1 generation. Plant ID No. of tested plants Genetic segregation in T 1 population for TIPS point mutations Wild type Heterozygote Homozygote Cas9-free* (%) T0-RP /47 (6.4) T0-RP /51 (13.7) T0-RP /38 (5.3) T0-RP /9 (0) T0-IP /28 (7.1) T0-IP /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. 20 NATURE PLANTS