Lettershttps://doi.org/10.1038/s41587-020-0581-5

Targeted, efficient sequence insertion and replacement in riceYuming Lu   1,4, Yifu Tian   1,4, Rundong Shen   1, Qi Yao   1, Mugui Wang   1, Mei Chen1, Jinsong Dong   1, Tongen Zhang   1, Feng Li2, Mingguang Lei   1 and Jian-Kang Zhu   1,3 ✉

1Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China. 2Shandong Shunfeng Biotechnology Co. Ltd, Jinan, China. 3Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA. 4These authors contributed equally: Yuming Lu, Yifu Tian. ✉e-mail: jkzhu@psc.ac.cn

SUPPLEMENTARY INFORMATION

In the format provided by the authors and unedited.

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1

Supplementary Information

Supplementary Item Page

Supplementary Figure 1. CRISPR/Cas9 vector and donor DNA. 2

Supplementary Figure 2. ADHE-inserted genomic sequences of SKC1, DRO1, SLR1 and

SOS1. 3

Supplementary Figure 3. Schematic overview of genotyping approach for targeted insertion

mutants. 4

Supplementary Figure 4. Genotyping of targeted insertion mutants at DRO1 using ssADHE,

dsADHE and modified ADHE. 5

Supplementary Figure 5. Sequences of the chemically modified donor DNA for targeted

insertion. 6

Supplementary Figure 6. Predicted donor-inserted genomic sequences of WARK71.1, DEP1,

BZR1, bZIP5, OsPAP10a, OsPAP10c, OsPAP21b and AVP1. 7

Supplementary Figure 7. Targeted insertion of UBI and CmYLC9.11 promoters into SKC1

and SLR1. 8

Supplementary Figure 8. Schematic overview of the TR-HDR method at SLR1. 9

Supplementary Figure 9. Genotyping of base-substituted mutants at SLR1 using TR-HDR. 10

Supplementary Figure 10. Sequences for base substitutions and flag tag fusion. 11

Supplementary Table 1. Summary of targeted loci in this study. 12

Supplementary Table 2. Sequencing results of T0 and T1 plants. 13-17

Supplementary Table 3. Insertion number of donors in transgenic lines determined using

qPCR. 18

Supplementary Table 4. Genotyping results of the offspring of the analyzed rice lines. 19

Supplementary Table 5. Primers used in this study. 20-22

Supplementary Table 6. Chemically modified oligos used in this study. 23

2

Supplementary Figure 1. CRISPR/Cas9 vector and donor DNA. (a) Schematic diagram of the

CRISPR/Cas9 vector. (b) Sequences of the ssADHE, dsADHE and chemically modified ADHE. Nucleotide

polymorphisms are indicated in red.

3

Supplementary Figure 2. ADHE-inserted genomic sequences of SKC1, DRO1, SLR1 and SOS1. ADHE

is indicated in yellow. Nucleotides in red, blue and bold are the 5’ UTR, coding region and targeting

sequences, respectively. Primer sequences are underlined.

4

Supplementary Figure 3. Schematic overview of genotyping approach for targeted insertion mutants.

(a,b) Schematic diagram of the forward and reverse insertion of ADHE in the genome. (c) Predicted length

of amplicons.

5

Supplementary Figure 4. Genotyping of targeted insertion mutants at DRO1 using ssADHE, dsADHE

and modified ADHE. Genomic DNA was amplified using primers flanking target sites (F+R). Forward (+,

middle) and reverse (-, lower) insertion mutants were identified using primers DRO1-F2 + ADHE-R and

DRO1-F2 + ADHE-F, respectively. Successful inserted mutants are indicated in bold. One biological

experiment was performed.

6

Supplementary Figure 5. Sequences of the chemically modified donor DNA for targeted insertion. “P”

and “*” stands for 5’ phosphorylation and phosphorothioate-linkage modification, respectively.

7

Supplementary Figure 6. Predicted donor-inserted genomic sequences of WARK71.1, DEP1, BZR1,

bZIP5, OsPAP10a, OsPAP10c, OsPAP21b and AVP1. Donor sequence is indicated in yellow. Nucleotides

in red, blue and bold are the 5’ UTR, coding region and targeting sequences, respectively. Primer

sequences are underlined.

8

Supplementary Figure 7. Targeted insertion of UBI and CmYLC9.11 promoters into SKC1 and SLR1.

(a,b) Schematic diagram of SKC1 and SLR1 with their sgRNA target (underlined) and PAM sequences

(bolded) in the 5’UTR (boxed). (c,d) Sanger sequencing chromatograms for transgenic lines SLR1-UBI#62,

SLR1-CMP#29, SKC1-UBI#28 and SKC1-CMP#49. Donor sequence is indicated in red. One biological

experiment was performed. (e) Transcript levels of SLR1 in T0 targeted insertion seedlings quantified

using qRT-PCR. Mutants containing forward donor sequences are marked in bold. ACT1 was used as

internal control. Transcript levels in the mutants were normalized to those of WT (wild type) control (n = 3

biologically independent experiments). All values represent means ± s.e.m. (f) Phenotypes of T0

transgenic seedlings of SLR1. One biological experiment was performed. WT, wild type. Scale bar, 10 cm.

9

Supplementary Figure 8. Schematic overview of the TR-HDR method at SLR1. (a) Chemically modified

dsODN (DN06) for TR-HDR. “P” and “*” stands for 5’ phosphorylation and phosphorothioate-linkage

modification, respectively. (b) Schematic overview of the TR-HDR process at SLR1. The sgRNA target

sites are boxed and tandem-repeat sequences are underlined. (c) Comparison between modified and wild

type genomic sequences. Nucleotide polymorphism is marked in red.

10

Supplementary Figure 9. Genotyping of base-substituted mutants at SLR1 using TR-HDR. (a) Schematic

diagram of the forwardly inserted dsODN at SLR1. (b) Predicted base-substituted sequences of SLR1.

Primers are underlined and substituted nucleotides are indicated in red. EcoRI recognition site is shown

in shadow. (c) Sequencing results of SLR1 mutants. Signs “d” and “i” indicate the number of nucleotides

deleted and inserted, respectively. Precisely substituted nucleotides are marked in red.

11

Supplementary Figure 10. Sequences for base substitutions and flag tag fusion. (a) Sequences of the

chemically modified donor DNA for TR-HDR. “P” and “*” stand for 5’ phosphorylation and

phosphorothioate-linkage modification, respectively. (b) Predicted sequence outcomes of TR-HDR for TT1,

NRT1.1b and UBQ6. Primers are underlined and substituted nucleotides or flag sequences are indicated

in red.

12

Supplementary Table 1. Summary of targeted loci in this study

Locus Msu ID Region Modification sgRNA Target sequence (5'-3')

SKC1 LOC_Os01g20160 5'UTR Insertion sgRNA-1 AACCCAAGAACGAAACACATAGG

DRO1 LOC_Os09g26840 5'UTR Insertion sgRNA-2 CCAGGATATGAAGGTAAAGGCTC

SLR1 LOC_Os03g49990 5'UTR Insertion sgRNA-3 ATTACTAGCTATAGCTAGGTAGG

SOS1 LOC_Os12g44360 5'UTR Insertion sgRNA-4 TCCTCCTCATCAGTAATCCATGG

WRKY71.1 LOC_Os02g08440 5'UTR Insertion sgRNA-11 AGTGATAAATCGTTCGCCGATGG

DEP1 LOC_Os09g26999 5'UTR Insertion sgRNA-12 GGCCGTTGGCGTGCTGCGTGTGG

BZR1 LOC_Os07g39220 5'UTR Insertion sgRNA-13 AGGTGAGGGACGGAGCTGGTGGG

bZIP5 LOC_Os05g49420 5'UTR Insertion sgRNA-14 AGACAGCAGGCTTTCATAAAAGG

OsPAP10a LOC_Os01g56880 Promoter Insertion sgRNA-15 CCGCAGAACAGTTCGAGTTGACC

OsPAP10c LOC_Os12g44020 Promoter Insertion sgRNA-16 CCACTGTGTTTCAAAGATGTAGC

OsPAP21b LOC_Os11g05400 Promoter Insertion sgRNA-17 CCGTTCAGACACGGCACGCATAA

AVP1 LOC_Os02g09150 Promoter Insertion sgRNA-18 CGTACTCCTCTTGCTCCGCTCGG

SLR1 LOC_Os03g49990 CDS Bases substitution sgRNA-6 CCTCGGACCTCTCCTCCTGGGTC

TT1 LOC_Os03g26970 CDS Bases substitution sgRNA-7 ACAGTATTATCGTCTGTACAAGG

NRT1.1b LOC_Os10g40600 CDS Bases substitution sgRNA-8 CGGCGACGGCGAGCAAGTGGAGG

UBQ6 LOC_Os02g06640 CDS Flag fusion sgRNA-9 CTGGTGCTCCGCCTCCGTGGTGG

TT1 LOC_Os03g26970 CDS Flag fusion sgRNA-10 CCGGCGATGGGCGACAGCCAGTA

13

Supplementary Table 2. Sequencing results of T0 and T1 plants

Mutant 5'junction -Donor- 3'junction

Junctions No.

* 5' 3'

DRO1-

ADHE

Ref …TTCTTCAGGCCAGGAGTGAATTCCAAGCAA……GAGCTTTCGATCTATTATGAAGGTAAAGGC… #40,T0 …TTCTTCAGGCCA---GAATTCCAAGCAA……GAGCTTTCGATCT-ATGAAGGTAAAGGC… d3 d1 8

#64,T0 …TTCTTCAGGCCAGGAGTGAATTCCAAGCAA……GAGCTTTCGATCTATgtgaattccaTATGAAGGTAAAGGC… 0 i10 5

…TTCTTCAGGCCAGGAGTGAATTCCAAGCAA……GAGCTTTCGATCTATgtgaatt…gagtgaTATGAAGGTAAAGGC… 0 i29 1

#79,T0 …TTCTTCAGGCCAGGAGTGAATTCCAAGCAA……GAGCTTTCGATCTATTATGAAGGTAAAGGC… 0 0 8

#81,T0 …TTCTTCAGGCCAG--GTGAATTCCAAGCAA……GAGCTTTCGATCTATcTATGAAGGTAAAGGC… d3 i1 8

SLR1-

ADHE

Ref …TACTAGCTATAGCTAGTGAATTCCAAGCAA……GAGCTTTCGATCTATGGTAGGTTTGGGGGA… #30,T0 …TACTAGCTATAGCT-GTGAATTCCAAGCAA……GAGCTTTCGATCTATGGTAGGTTTGGGGGA… d1 0 6

#34,T0 …TACTAGCTATAGCTAtGTGAATTCCAAGCAA……GAGCTTTCGATCTAT---AGGTTTGGGGGA… i1 d3 4

…TACTAGCTATAGCTAGTGAATTCCAAGCAA……GAGCTTTCGATCTAT---AGGTTTGGGGGA… 0 d3 2

#37,T0 …TACTAGCTATAGCT-GTGAATTCCAAGCAA……GAGCTTTCGATCTATGGTAGGTTTGGGGGA… d1 0 6

SOS1-

ADHE

Ref …CTCCTCATCAGTAATGTGAATTCCAAGCAA……GAGCTTTCGATCTATCCATGGACAATCCCG… #57,T0 …CTCCTCATCAGTAATGTGAATTCCAAGCAA……GAGCTTTCGATCTATCCATGGACAATCCCG… 0 0 5

#64,T0 …CTCCTCATCAGTAA--TGAATTCCAAGCAA……GAGCTTTCGATCTATCCATGGACAATCCCG… d2 0 3

#65,T0 …CTCCTCATCAGTAA--TGAATTCCAAGCAA……GAGCTTTCGATCTATCCATGGACAATCCCG… d2 0 6

SKC1-

ADHE

Ref …CCCAAGAACGAAACAGTGAATTCCAAGCAA……GAGCTTTCGATCTATCATAGGAGAGAAATG… #49,T0 …CCCAAGAACGAAACAGTGAATTCCAAGCAA……GAGCTTTCGATCTATCATAGGAGAGAAATG… 0 0 6

#52,T0 …CCCAAGAACGAAACAcGTGAATTCCAAGCAA……GAGCTTTCGATCTATCATAGGAGAGAAATG… i1 0 6

#57,T0 …CCCAAGAACGAAACAcGTGAATTCCAAGCAA……GAGCTTTCGATCTATCATAGGAGAGAAATG… i1 0 5

WRKY-

AMVE

Ref …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… #11,T0 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 4

#16,T0 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 4

#16-1,T1 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 4

#16-2,T1 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 4

#16-3,T1 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 2

#16-4,T1 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 4

#16-5,T1 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 3

#16-6,T1 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 4

#16-7,T1 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 4

#16-8,T1 …TCAGTGATAAATCGTTCGCATAGATACCTCGAG……TCCATCCCTTTTGCCGATGGATCCGTG… 0 0 4

BZR1-

AMVE

Ref …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCGGTGGGTATGAC… #02,T0 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCtGGTGGGTATGAC… 0 i1 3

#03,T0 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCtGGTGGGTATGAC… 0 i1 4

#10,T0 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCGGTGGGTATGAC… 0 0 4

#11,T0 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCGGTGGGTATGAC… 0 0 4

#22,T0 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTG-tGGTGGGTATGAC… 0 d1i1 2

#29,T0 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGC--TGGGTATGAC… 0 d2 1

…GGGAGGTGAGGGACGG----ATAGATACCTCGAG……TCCATCCCTTTTGC--TGGGTATGAC… d4 d2 3

14

#31,T0 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCGGTGGGTATGAC… 0 0 4

#31-3,T1 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCGGTGGGTATGAC… 0 0 4

#31-4,T1 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCGGTGGGTATGAC… 0 0 4

#31-8,T1 …GGGAGGTGAGGGACGGAGCTATAGATACCTCGAG……TCCATCCCTTTTGCGGTGGGTATGAC… 0 0 3

#41,T0 …GGGAGGTGAGGGACGGAGC-ATAGATACCTCGAG……TCCATCCCTTTTGCtGGTGGGTATGAC… d1 i1 4

#55,T0

…GGGAGGTGAGGGACGGAGC-ATAGATACCTCGAG……TCCATCCCTTTTGC-GTGGGTATGAC… d1 d1 2

…GGGAGGTGAGGGACGG-------GATACCTCGAG……TCCATCCCTTTTGC-GTGGGTATGAC… d7 d1 1

…GGGAGGTGAGGGACGG-------GATACCTCGAG……TCCATCCCTTTTGCtGGTGGGTATGAC… d7 i1 1

DEP1-

AMVE

Ref …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… #6,T0 …GGCCGTTGGCGTGCTGC-TAGATACCTCGAG……TCCATCCCTTTTGCtGTGTGGAGATGGGGGA… d1 i1 4

#7,T0 …GGCCGTTGGCGTGCTGC-TAGATACCTCGAG……TCCATCCCTTTTGCtGTGTGGAGATGGGGGA… d1 i1 4

#8,T0 …GGCCGTTGGCGTGCTGC-TAGATACCTCGAG……TCCATCCCTTTTGCtGTGTGGAGATGGGGGA… d1 i1 3

#15,T0 …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… 0 0 4

#21,T0 …GGCCGTTGGCGTGCTGCggtat---------……TCCATCCCTTTTGC--GTGGAGATGGGGGA… i5d20 d2 1

…GGCCGTTGGCGTGCTGCggtat---------……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… i5d20 0 2

#22,T0 …GGCCGTTGGCGTGCTGCggtat---------……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… i5d20 0 4

#23,T0 …GGCCGTTGGCGTGCT--ATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… d2 0 2

#25,T0 …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… 0 0 4

#26,T0 …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… 0 0 4

#27,T0 …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… 0 0 3

#29,T0 …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCcGTGTGGAGATGGGGGA… 0 i1 4

…GGCCGTTGGCGTGCTG-ATAGATACCTCGAG……TCCATCCCTTTTGCcGTGTGGAGATGGGGGA… d1 i1 4

#47,T0 …GGCCGTTGGCGTGCTG-ATAGATACCTCGAG……TCCATCCCTTTTG-tGTGTGGAGATGGGGGA… d1 d1i1 3

#48,T0 …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… 0 0 4

#55,T0 …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTT--cGTGTGGAGATGGGGGA… 0 d2i1 4

#56,T0 …-----------------------ACCTCGAG……TCCATCCCTTTTGC--GTGGAGATGGGGGA… d45 d2 1

…GGCCGTTGGCG------------ACCTCGAG……TCCATCCCTTTTGC--GTGGAGATGGGGGA… d12 d2 3

#59,T0 …GGCCGTTGGCG----------ATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… d10 0 2

…GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… 0 0 2

#60,T0 …GGCCGTTGGCG----------ATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… d10 0 3

#64,T0 …GGCCGTTGGCGTGCTGCATAGATACCTCGAG……TCCATCCCTTTTGCGTGTGGAGATGGGGGA… 0 0 4

#72,T0 …GGCCGTTGGCGTGCTGC-TAGATACCTCGAG……TCCATCCCTTTTGCtGTGTGGAGATGGGGGA… d1 i1 4

#73,T0 …GGCCGTTGGCGTGCTGC-TAGATACCTCGAG……TCCATCCCTTTTGCtGTGTGGAGATGGGGGA… d1 i1 4

bZIP5-

AMVE

Ref …AGACAGCAGGCTTTCATATAGATACCTCGAG……TCCATCCCTTTTGCAAAAGGCATGGCTCAT…

#17,T0 …AGACAGCAGGCTTTCAT-TAGATACCTCGAG……TCCATCCCTTTTGC------------TCAT… d1 d13 4

…AGACAGCAGGCTTTCATATAGATACCTCGAG……TCCATCCCTTTTGC------------TCAT… 0 d13 3

#18,T0 …AGACAGCAGGCTTTCAT-TAGATACCTCGAG……TCCATCCCTTTTGCcAAAAGGCATGGCTCAT… d1 i1 4

#31,T0 …AGACAGCAGGCTTTCA-ATAGATACCTCGAG……TCCATCCCTTTTGCAAAAGGCATGGCTCAT… d1 0 4

SLR1-

CMP

Ref …ACTAGCTATAGCTAGCCAGGGTTTTCCCAGTCAC……GAGCTAAGCTAACAAAGCACGGTAGGTTTGGGGGAGG… #09,T0 …ACTAGCTAT----gGCCAGGGTTTTCCCAGTCAC……GAGCTAAGCTAACAAAGCA---------------AGG… d5i1 d15 6

#17,T0 …--------------------------CCAGTCAC……GAGCTAAG----CAAAGCACGGTAGGTTTGGGGGAGG… d69 d4 4

…--------------------------CCAGTCAC……GAGCTAAG----------------------GGGGAGG… d69 d22 1

15

#29,T0 …ACTAGCTATAGCT-GCCAGGGTTTTCCCAGTCAC……GAGCTAAGCTAACAAAGCAtatgTCATGAAGCGCGA… d1 d18i4 6

SLR1-

UBI

Ref …ACTAGCTATAGCTAGCCAGGGTTTTCCCAGTCAC……CTTCTGCAGATTCAGAGCTGGTAGGTTTGGGGGAGGCGAG… #29,T0 …----------------------------------……CTTCTGCAGATTCc-----------TTTGGGGGAGGCGAG… d138 i1d12 5

#62,T0 …ACTAGCTATAGCctt-CAGGGTTTTCCCAGTCAC……CTTCTGCAGAT---------------TTGGGGGAGGCGAG… d4i3 d15 6

SKC1-

CMP

Ref …CAAGAACGAAACAGCCAGGGTTTTCCCAGTCAC……GAGCTAAGCTAACAAAGCACCATAGGAGAGAAATGAG… #14,T0 …CAAGAACGA----GCCAGGGTTTTCCCAGTCAC……GAGCTAAGCTAACAAAGCAC-ATAGGAGAGAAATGAG… d4 d1 6

#16,T0 …CAAGAAC--------tgGGGTTTTCCCAGTCAC……GAGCTAAGCTAACAAAGCAC-ATAGGAGAGAAATGAG… d10i2 d1 6

#28,T0 …CAAGAACGAAAC--CCAGGGTTTTCCCAGTCAC……GAGCTAAGCTAACAAAGCAC-ATAGGAGAGAAATGAG… d2 d1 4

…CAAGAACGAAAC--CCAGGGTTTTCCCAGTCAC……----------------------------ccAAATGAG… d2 d110i2 1

#49,T0 …CAAGAACGA----GCCAGGGTTTTCCCAGTCAC……GAGCTAAGCTAACAAA---------------------… d4 d21 5

SKC1-

UBI

Ref …CAAGAACGAAACAGCCAGGGTTTTCCCAGTCAC……CTTCTGCAGATTCAGAGCTCATAGGAGAGAAATGAG… #25,T0 …CAAGAACG------CCAGGGTTTTCCCAGTCAC……CTTCTGCAGATTCAGAGC-----------AAATGAG… d6 d11 6

#28,T0 …CAAGAACGAAACA--------------------……CTTCTGCAGATTCAGAGCTattaCATAGGAGAGAAATGAG… d28 i4 3

TT1-BS

WT …CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCGTCTGTACAAGGTATTTGATG… Ref …CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCACCTCTACAAGGTATTTGATG…

#03,T0

…CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCACCTCTACAAGGTATTTGATG… 0 0 3

…CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCGTCTG-ACAAGGCCTTGTTTT……AGTATTATCACCTCTACAAGGTATTTGATG… repeat 4

…CATGTGCTTACCTTGTTTTTTTTTT……AGTATTAT-------ACAAGGCCTTGTTTT……AGTATTATCACCTCTACAAGGTATTTGATG… repeat 1

#25,T0

…CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCACCTCTACAAGGTATTTGATG… 0 0 1

…CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCGTCTG-ACAAGGTATTTGATG… indel 4

…CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATC-----------GTATTTGATG… indel 1

…CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCGTCTG-ACAAGGCCTTGTTTT……AGTATTATCACCTCTACAAGGTATTTGATG… repeat 1

#52,T0 …CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCACCTCTACAAGGTATTTGATG… 0 0 2

…CATGTGCTTACCTTGTTTTTTTTTT……AGTATTATCGTCTG-ACAAGGTATTTGATG… indel 5

NRT-BS

WT …ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGAGCAAGTGGAGGCTGTGCACG… Ref …ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAAATGGAGGCTGTGCACG…

#08,T0 …ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAAATGGAGGCTGTGCACG… 0 0 3

…ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGAGCAA--GGAGGACGTGTTTG……GCGATGGCGAGTAAATGGAGGCTGTGCACG… repeat 5

#46,T0

…ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGAG-------AGGCTGTGCACG… indel 3

…ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAAATGGAGGCTGTGCACG… 0 0 1

…ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAAA----GGCTGTGCACG… 0 d4 1

…ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGA-----TGGAGGACGTGTTTG……GCGATGGCGAGTAAA----GGCTGTGCACG… repeat 1

…ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGAGCAAG-GGAGGACGTGTTTG……GCGATGGCGAGTAAATGGAGGCTGTGCACG… repeat 2

#57,T0

…ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAAATGGAGGCTGTGCACG… 0 0 2

…ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGAGC-AGTGGAGGCTGTGCACG… indel 4

…ATAATCATTGACGTGTTTGGTTTGT……-----d26-------TGGAGGACGTGTTTG……GCGATGGCGAGTAAATGGAGGCTGTGCACG… repeat 1

#58,T0 …ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAAATGGAGGCTGTGCACG… 0 0 2

…ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGAGC-AGTGGAGGCTGTGCACG… indel 6

#62,T0 …ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAAATGGAGGCTGTGCACG… 0 0 4

#81,T0

…ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGAGCAAGcTGGAGGCTGTGCACG… indel 3

…ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAAATGGAGGCTGTGCACG… 0 0 2

…ATAATCATTGACGTGTTTGGTTTGT……GCGATGGCGAGTAA-TGGAGGCTGTGCACG… 0 d1 1

16

…ATAATCATTGACGTGTTTGGTTTGT……GCGACGGCGAGCAAG-GGAGGACGTGTTTG……GCGATGGCGAGTAAATGGAGGCTGTGCACG… repeat 2

UBQ6-

Flag

WT …ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCCGTGGTGGTCAGTAATC… Ref …ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAGTGGTGGTCAGTAATC…

#8,T0

…ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAG--GTGGTCAGTAATC… 0 d2 3

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCCGaTGGTGGTCAGTAATC… indel 9

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCC-TGGTGGAGGA……CGATAAGTAGCTGAG--GTGGTCAGTAATC… repeat 4

#9,T0 …ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAG--GTGGTCAGTAATC… 0 d2 2

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCCGaTGGTGGTCAGTAATC… indel 5

#14,T0

…ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAGattGGTGGTCAGTAATC… 0 d1i3 1

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCCGaTGGTGGTCAGTAATC… indel 5

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCC----TGGTGGAGGA……CGATAAGTAGCTGAG-GGTGGTCAGTAATC… repeat 1

#48,T0

…ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAG------TCAGTAATC… 0 d6 1

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCC-TGGTGGTCAGTAATC… indel 6

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCC-TGGTGGAGGA……CGATAAGTAGCTGAGTGGTGGTCAGTAATC… repeat 1

#59,T0

…ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAGTGGTGGTCAGTAATC 0 0 2

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCC-TGGTGGAGGA……CGATAAGTAGCTGAGTGGTGGTCAGTAATC… repeat 4

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCT------------------TGGAGGA……CGATAAGTAGCTGAGTGGTGGTCAGTAATC… repeat 2

#64,T0

…ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAGTGGTGGTCAGTAATC… 0 0 2

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCCGaTGGTGGTCAGTAATC… indel 5

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCC---GTGGAGGA……CGATAAGTAGCTGAG--GTGGTCAGTAATC… repeat 1

#65,T0 …ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAGTGGTGGTCAGTAATC… 0 0 2

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCCGaTGGTGGTCAGTAATC… indel 6

#81,T0 …ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAGagTGGTGGTCAGTAATC… 0 i2 2

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCCGaTGGTGGTCAGTAATC… indel 5

#90,T0

…ACTACAACATCCAGAAGGAGTCCACCCTCC……CGATAAGTAGCTGAG--GTGGTCAGTAATC… 0 d2 2

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCC---GTGGTCAGTAATC… indel 4

…ACTACAACATCCAGAAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCC----TGGAGGA……CGATAAGTAGCTGAG---TGGTCAGTAATC… repeat 1

TT1-

Flag

WT …GAGCCGCCACCGGCGATGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… Ref …GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT…

#22,T0 …GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… d1 0 4

…GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 4

#42,T0

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… 0 0 1

…GAGCCGCCACCGGC-----GCGACAGCCAGTACTCCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… indel 5

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAaagaGGTGCGCTACCCTCT… repeat 2

#45,T0

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… 0 0 2

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 4

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCT----TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 1

#66,T0

…GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… d1 0 1

…GAGCCGCCACCG--aGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… d3i1 0 1

…GAGCCGCCACC--------GCGACAGCCAGTACTCCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… indel 4

…GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 1

17

#67,T0 …GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… d1 0 1

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCT----TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 5

#68,T0 …GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… d1 0 3

…GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTT-------CGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 5

#80,T0

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… 0 0 2

…GAGCCGCCACCGGCGaATGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… indel 4

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 2

#82,T0

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… 0 0 2

…GAGCCGCCACCGGCGaATGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… indel 2

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 3

#89,T0

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… 0 0 2

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAt------------AGTACTCCTTCTCCCTCACCACCTT… repeat 2

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 3

#91,T0

…GAGCCGCCACC---tGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… d4i1 0 2

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAt------------AGTACTCCTTCTCCCTCACCACCTT… repeat 1

…GAGCCGCCACCGGCGGACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 3

#98,T0

…GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… d1 0 2

…GAGCCGCCACCGGCG---GGCGACAGCCAGTACTCCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… indel 3

…GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 3

#105,T0

…GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… d1 0 1

…GAGCCGCCACCGGCG---GGCGACAGCCAGTACTCCTTCTCCCTCACCACCTTCAGGTGCGCTACCCTCT… indel 4

…GAGCCGCCACCGGC-GACCTCAAGATGGAC……CCTTCTCCCTCACCACCTTCA-TGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTT… repeat 3

Signs “d” and “i” indicate the number of nucleotides deleted and inserted, respectively. “0” means no indels detected. Sequences of donors are marked with an

underline. WT stands for wild type. Ref means the expected sequences as reference. * No. is the number of sequenced colonies.

18

Supplementary Table 3. Insertion number of donors in transgenic lines determined using qPCR

Mutant Generation DNA used for 50 µl gold microparticles Copy number

Donor CRISPR/Cas9 plasmid Donor Cas9

SKC1-ADHE#50-1

T1 ADHE 10

pmol 0.1 pmol

9 2

SKC1-ADHE#50-2 8 2

SKC1-ADHE#50-3 10 0

SKC1-ADHE#58-1 8 9

SKC1-ADHE#58-2 6 5

SKC1-ADHE#58-3 8 6

SKC1-ADHE#58-4 10 8

CMP-KI#09-1

T1 CmYLCV9.11 2 pmol 0.05 pmol

4 2

CMP-KI#09-2 4 4

CMP-KI#09-3 2 0

CMP-KI#09-4 5 1

CMP-KI#09-5 3 2

CMP-KI#09-6 4 1

CMP-KI#09-7 7 3

CMP-KI#09-8 2 0

UBQ-Flag#8-1

T1 DN09 2 pmol 0.05 pmol

2 2

UBQ-Flag#8-2 4 3

UBQ-Flag#8-3 3 0

UBQ-Flag#8-4 2 4

UBQ-Flag#8-5 3 0

UBQ-Flag#14-1 5 2

UBQ-Flag#14-2 4 2

TT1-Flag#68-1

T1 DN10 2 pmol 0.05 pmol

3 5

TT1-Flag#68-2 7 4

TT1-Flag#68-3 2 0

TT1-Flag#68-4 4 4

TT1-Flag#68-5 9 2

TT1-Flag#68-6 5 5

TT1-Flag#68-7 5 3

TT1-Flag#68-8 3 1

TT1-Flag#68-9 3 1

TT1-Flag#68-10 4 5

TT1-Flag#68-11 3 4

TT1-Flag#68-12 2 1

TT1-Flag#68-13 3 5

TT1-Flag#68-14 6 4

TT1-Flag#68-15 6 4

TT1-Flag#82-1 4 0

19

Supplementary Table 4. Genotyping results of the offspring of the analyzed rice lines

Type Locus Modification

type

Parent Progeny

Name Generation Genotype Total Edited* Transmission

Targeted

insertion

DRO1 ADHE

insertion

DRO1-ADHE#77 T0 Heterozygous / Chimeric 23 13 56.50%

DRO1-ADHE#81 T0 Heterozygous / Chimeric 8 3 37.50%

DRO1-ADHE#77-1 T1 Heterozygous 94 73 77.70%

SKC1 ADHE

insertion

SKC1-ADHE#50 T0 Homozygous 21 21 100.00%

SKC1-ADHE#58 T0 Heterozygous / Chimeric 42 20 47.60%

SKC1-ADHE#50-1 T1 Homozygous 48 48 100.00%

SKC1-ADHE#58-3 T1 Heterozygous 86 63 73.30%

SKC1-ADHE#50-1-1 T2 Homozygous 48 48 100.00%

WRKY7

1.1 AMVE

insertion

WRKY-AMVE#04 T0 Heterozygous / Chimeric 47 25 53.20%

WRKY-AMVE#16 T0 Homozygous 46 46 100.00%

BZR1 BZR1-AMVE#22 T0 Homozygous 48 48 100.00%

BZR1-AMVE#31 T0 Heterozygous / Chimeric 44 20 45.50%

TR-HDR

TT1

Base

substitution

TT1-BS#03 T0 Heterozygous / Chimeric 48 12 25.00%

TT1-BS#25 T0 Heterozygous / Chimeric 62 2 3.20%

NRT1.1b

NRT-BS#08 T0 Heterozygous / Chimeric 48 18 37.50%

NRT-BS#46 T0 Heterozygous / Chimeric 47 3 6.40%

NRT-BS#57 T0 Heterozygous / Chimeric 48 0 0.00%

NRT-BS#62 T0 Homozygous 7 7 100.00%

UBQ

Flag tag

fusion

UBQ6-Flag#08 T0 Heterozygous / Chimeric 8 5 62.50%

UBQ6-Flag#14 T0 Heterozygous / Chimeric 45 2 4.40%

UBQ6-Flag#48 T0 Heterozygous / Chimeric 48 0 0.00%

TT1

TT1-Flag#42 T0 Heterozygous / Chimeric 47 0 0.00%

TT1-Flag#68 T0 Heterozygous / Chimeric 48 15 31.30%

TT1-Flag#82 T0 Heterozygous / Chimeric 42 1 2.40%

* Progenies that contain the expected modifications shown in Supplementary table 2, with or without off-target insertions of donors or

construct sequences were counted as edited.

20

Supplementary Table 5. Primers used in this study

Name Sequence(5'-3') Experiment

sgRNA1-UP TGTGTGACCCAAGAACGAAACACAT

Construction of

CRISPR/Cas9 plasmids

sgRNA1-LW AAACATGTGTTTCGTTCTTGGGTCA

sgRNA2-UP TGTGTGAGCCTTTACCTTCATATCC

sgRNA2-LW AAACGGATATGAAGGTAAAGGCTCA

sgRNA3-UP TGTGTGTTACTAGCTATAGCTAGGT

sgRNA3-LW AAACACCTAGCTATAGCTAGTAACA

sgRNA4-UP TGTGTGCCTCCTCATCAGTAATCCA

sgRNA4-LW AAACTGGATTACTGATGAGGAGGCA

sgRNA6-UP TGTGTGACCCAGGAGGAGAGGTCCG

sgRNA6-LW AAACCGGACCTCTCCTCCTGGGTCA

sgRNA7-UP TGTGTGCAGTATTATCGTCTGTACA

sgRNA7-LW AAACTGTACAGACGATAATACTGCA

sgRNA8-UP TGTGTGGGCGACGGCGAGCAAGTGG

sgRNA8-LW AAACCCACTTGCTCGCCGTCGCCCA

sgRNA9-UP TGTGTGTGGTGCTCCGCCTCCGTGG

sgRNA9-LW AAACCCACGGAGGCGGAGCACCACA

sgRNA10-UP TGTGTGACTGGCTGTCGCCCATCGC

sgRNA10-LW AAACGCGATGGGCGACAGCCAGTCA

sgRNA11-UP TGTGTGGTGATAAATCGTTCGCCGA

sgRNA11-LW AAACTCGGCGAACGATTTATCACCA

sgRNA12-UP TGTGTGGCCGTTGGCGTGCTGCGTG

sgRNA12-LW AAACCACGCAGCACGCCAACGGCCA

sgRNA13-UP TGTGTGGGTGAGGGACGGAGCTGGT

sgRNA13-LW AAACACCAGCTCCGTCCCTCACCCA

sgRNA14-UP TGTGTGGACAGCAGGCTTTCATAAA

sgRNA14-LW AAACTTTATGAAAGCCTGCTGTCCA

sgRNA15-UP TGTGTGGGTCAACTCGAACTGTTCTG

sgRNA15-LW AAACCAGAACAGTTCGAGTTGACCCA

sgRNA16-UP TGTGTGGCTACATCTTTGAAACACAG

sgRNA16-LW AAACCTGTGTTTCAAAGATGTAGCCA

sgRNA17-UP TGTGTGTTATGCGTGCCGTGTCTGAA

sgRNA17-LW AAACTTCAGACACGGCACGCATAACA

sgRNA18-UP TGTGTGCGTACTCCTCTTGCTCCGCT

sgRNA18-LW AAACAGCGGAGCAAGAGGAGTACGCA

SKC1-NGSF CCCACATACAGTGATCACACA Used for NGS

SKC1-NGSR TACAGCAGTTGGAGCCAGAA

SKC1-F ACATGGCTTCCTAATTCCTA

Genotype of ADHE insertion SKC1-R GTCATATCTAGGAGTAGTGGCA

SKC1-F2 TGGCTAGTAGTGTTCATCAGAT

DRO1-F CCATTGCTAGGAGCTTGCATTTC

21

DRO1-R ATGATTAATCACACCGATGGTTTGAGCCTT

DRO1-F2 TGTTATCATTCCATGGCTCA

SLR1-F CAAAGCCGAAACCGAGGAGA

SLR1-R CATCACCTTGTCCTTGCACG

SLR1-F2 GTAACTGGATGGTGAGGTCTGT

SOS1-F TGCCTTGGATTTTCCACTTGCA

SOS1-R ACGAAGAGCACCGCGTCAT

SOS1-F2 AGGTTCGGTCGAATTGGGTGTA

ADHE-F GAATTCCAAGCAACGAACTG

ADHE-R AGATCGAAAGCTCAGGTTTT

PWRKY-F TTGGCTCAGCTTGACATCGT

Genotype of AMVE insertion

PWRKY-R AGCACCTTGGGCTTAACCATG

PWRKY-F2 CCCTCCAGTCTCCTTCCGATT

PDEP-F CTGCTGCTGCTATAATGCCCA

PDEP-R TCCGGGTACCTCGGCGGCGAC

PDEP-F2 GCTGTTGAACTCTCTCTTCCC

PBZR-F TGGAGAAAGGAGAGGTGGAGG

PBZR-R TGTTGTTCTCCCTCTCCTTCC

PBZR-F2 GAAAACGTGGGTCCGAAGGTG

PbZIP-F TAGTTGCTTAACTGCACACCA

PbZIP-R TTAGGAGGGGAGGTTGTTTTC

PbZIP-F2 GAATATCTAGTTACTGAAGCA

AMVF-R GTATTTGAAAGAAAATTAAAA

AMVR-R TTATTTTTAATTTTCTTTCAA

P1BSF-R TCAGTATCTTCCTTAGCATATTCT

Genotype of P1BS insertion

P1BSR-R GTAGAATATGCTAAGGAAGATACT

OsPAP10a-F2 CAAATTAATTTCATGTGATGTGCA

OsPAP10a-F TCGCGAATCATCAAACCGAA

OsPAP10a-R GAGTGTTGTGGCGATCTGGA

OsPAP10c-F2 ATGAAAGTAGCTAGACTGCTCCTA

OsPAP10c-F AATCGACCAAAAGGTGGCAG

OsPAP10c-R TGCACATACTGTTACTTGCCCA

OsPAP21b-F2 TCCAGTACTGTGGCCACCGA

OsPAP21b-F CATGCTGCCAGACGAGAGTAA

OsPAP21b-R AACAAAGTGAACGCCCGATG

AVP1-F2 AGACATGCCCCACAATCAGG

AVP1-F GCCACCAAGAAAATCCCCAAA

AVP1-R CTCCTCACAATTCGCGCC

PSKC-F4 GACGTCGACGCAGACACTTGTAG

Genotype of UBI and

CmYLCV9.11 insertion

PSKC-R4 CGGCACCATGCTGGTCTTCATC

CmpKI-F GTCCACCATGTGAAGGCATC

CmpKI-R AGCCCTTACGTCATTGCTCC

PSLR-F4 GCCTTCCTCTCTGATCACCTG

22

PSLR-R4 CATCACCTTGTCCTTGCACG

UBIKI-F CTAGGATAGGTATACATGTTGATGCGG

UBIKI-R AGATAAAACTGTAGAGTCCTGTTGTC

SLR-BS-F1 ATGGCCGACGTCGCGCAGAA

Base substitutions

SLR-BS-R1 AGTCAGCAGCCGACGGGTCA

SLR-BS-R2 GCTCTCGACCCAACTGAATT

NRT-BS-F1 ACTTTGGCTGCCAAGAAACACCACA

NRT-BS-R1 CGTAGAAGGGGACGGTGAGGAGGAT

NRT-BS-F2 GGCGATGGCGAGTAAATG

TT1-BS-F1 GAAATGCTTATTACAAGTGCCAACT

TT1-BS-R1 TCCTGCATGACAGCAGCAGTTTCTC

TT1-BS-F2 CAACAGTATTATCACCTCTAC

FLAG-F CTATAAGGACCACGACGGAGACTACAAG

Flag fusion

FLAG-R TCGTCATCGTCTTTGTAATCAATATCATGATCC

UBQ6-Flag-R1 CGGACTCGATGGTCCATTAAACCAGACACA

UBQ6-Flag-F1 GATCTTTGTCAAGACCCTCACA

TT1-Flag-F1 CGAGTCGGCGGCGACAACAAATCGA

TT1-Flag-R1 AGCCTAACCTACCCACCCTCTGCGA

ACT1-qF GACCCAGATCATGTTTGAGACC

qRT-PCR for SLR1 and

SKC1 expression

ACT1-qR CATCACCAGAGTCCAACACAATAC

SLR1-qF AGCAGGTGGGTTGGAAGCTT

SLR1-qR AGCATGAACGGCTCCAAGTC

SKC1-qF TGAGATGGTGCTCGACCTCT

SKC1-qR TGGACAGGTACGAAAGTGGT

ADH-qF ATTCCAAGCAACGAACTGCGA

Identification of donor

insertion number

ADH-qR GATCGAAAGCTCAGGTTTTCTT

SPSF-qF TTGCGCCCTGACGGATAT

SPSF-qR CGGTTGATCTTTTCGGGATG

Hyg-qF CTATTTCTTTGCCCTCGGACGA

Hyg-qR GGACCGATGGCTGTGTAGAAG

Cmp-qF CAATAATGGAGCAATGACGTAAGG

Cmp-qR TGACGGGTGAGTGGACATTTC

Flag-qF GACTATAAGGACCACGACGG

Flag-qR ATCGTCATCGTCTTTGTAATCA

SPSF-PF AATTAGAGGAGTCCGTGGCTT

SPSF-PR GCATGTGTGCATATATACGTGTG

ADH-PF CCTCCACCTCCTCGATCGACAG

ADH-PR CTTCACTCTGCAATGCATCATCATCC

Hyg-PF CATCCGGAGCTTGCAGG

Hyg-PR CAACTTAATAACACATTGCGGAC

23

Supplementary Table 6. Chemically modified oligos used in this study

Name Sequence (5'-3') Experiment

ADHE-ssODN GAATTCCAAGCAACGAACTGCGAGCGTTCAAGAAAAAAGAAAACCTGAGCTTTCGATCT

ADHE donor

ADHE-dsODN-UP GAATTCCAAGCAACGAACTGCGAGATTTCAAGAAAAAAGAAAACCTGAGCTTTCGATCT

ADHE-dsODN-LW AGATCGAAAGCTCAGGTTTTCTTTTTTCTTGAAATCTCGCAGTTCGTTGCTTGGAATTC

ADHE-UP P_G*T*GAATTCCAAGCAACGAACTGCGAGTGATTCAAGAAAAAAGAAAACCTGAGCTTTCGATCT*A*T

ADHE-LW P_A*T*AGATCGAAAGCTCAGGTTTTCTTTTTTCTTGAATCACTCGCAGTTCGTTGCTTGGAATTC*A*C

AMVE-UP P_A*T*AGATACCTCGAGTTTTTATTTTTAATTTTCTTTCAAATACTTCCATCCCTTTT*G*C AMVE donor

AMVE-LW P_G*C*AAAAGGGATGGAAGTATTTGAAAGAAAATTAAAAATAAAAACTCGAGGTATCT*A*T

P1BS-UP P_G*T*AGAATATGCTAAGGAAGATACT*G*A P1BS donor

P1BS-LW P_T*C*AGTATCTTCCTTAGCATATTCT*A*C

PUV4-PTF P_G*C*CAGGGTTTTCCCAGTCACGACGTTGTAA Amplify UBI and

CmYLCV9.11 CMP911-PTR P_G*T*GCTTTGTTAGCTTAGCTCTTACCTGTTT

UBI-PTR P_A*G*CTCTGAATCTGCAGAAGTAACACCAAAC

DN06-UP P_A*A*TTCAGTTGGGTCGAGAGCATGCTTTCCGAGCTCAACGCGCCGCTGCCCCCTATCCCGCCAGC

GCCGCCGGCTGCCCGCCATGCTTCCACCTC*G*G SLR1 substitution

DN06-LW P_C*C*GAGGTGGAAGCATGGCGGGCAGCCGGCGGCGCTGGCGGGATAGGGGGCAGCGGCGCGTT

GAGCTCGGAAAGCATGCTCTCGACCCAACTGAA*T*T

DN07-UP P_A*C*AAGGCCTTGTTTTTTTTTTCTTCCAGTGGCATGGGGCCAGACTTCCGTGTTTTAGTGAGGAAAA

GTCGAAAGCAAGCACAACAGTATTATCACCT*C*T TT1 substitution

DN07-LW P_A*G*AGGTGATAATACTGTTGTGCTTGCTTTCGACTTTTCCTCACTAAAACACGGAAGTCTGGCCCCA

TGCCACTGGAAGAAAAAAAAAACAAGGCCTT*G*T

DN08-UP P_T*G*GAGGACGTGTTTGGTTTGTAATATGGGTGCAGGTTCCTGGACCATGCGGCGATCATCGACAG

GTCGGCGGCGGAGTCGCCGGCGATGGCGAGTA*A*A NRT1.1b

substitution DN08-LW

P_T*T*TACTCGCCATCGCCGGCGACTCCGCCGCCGACCTGTCGATGATCGCCGCATGGTCCAGGAAC

CTGCACCCATATTACAAACCAAACACGTCCTC*C*A

DN09-UP P_T*G*GTGGAGGAGTCCACCCTCCACCTGGTGCTCCGCCTCCGTGGAGCACAGGGATCCGACTATAA

GGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGTAGCTG*A*G Flag tag fusion to

UBQ6 DN09-LW

P_C*T*CAGCTACTTATCGTCATCGTCTTTGTAATCAATATCATGATCCTTGTAGTCTCCGTCGTGGTCC

TTATAGTCGGATCCCTGTGCTCCACGGAGGCGGAGCACCAGGTGGAGGGTGGACTCCTCCAC*C*A

DN10-UP P_G*A*CCTCAAGATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGA

CGATGACGATAAGGGATCCATGGGCGACAGCCAGTACTCCTTCTCCCTCACCACCTTCACCGG*C*G Flag tag fusion to

TT1 DN10-LW

P_C*G*CCGGTGAAGGTGGTGAGGGAGAAGGAGTACTGGCTGTCGCCCATGGATCCCTTATCGTCATC

GTCTTTGTAATCAATATCATGATCCTTGTAGTCTCCGTCGTGGTCCTTATAGTCCATCTTGAGG*T*C