highly efficient dna-free plant genome editing using ...10.1038...lineage of crispr/cas9-induced...

Lettershttps://doi.org/10.1038/s41477-020-0704-5

Highly efficient DNA-free plant genome editing using virally delivered CRISPR–Cas9Xiaonan Ma1, Xiaoyan Zhang1, Huimin Liu1 and Zhenghe Li 1,2,3 ✉

1State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China. 2Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou, China. 3Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Zhejiang University, Hangzhou, China. ✉e-mail: [email protected]

SUPPLEMENTARY INFORMATION

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SUPPLEMENTARY MATERIALS

Table of Contents

Supplementary Methods

Supplementary References

Supplementary Figure 1. Sequences of the gRNA and Cas9 expression cassettes in the

SYNV-tgtRNA-Cas9 (a) and SYNV-gRNA-Cas9 (b) vectors.

Supplementary Figure 2. Schematic representation of transcription and processing of the

tgtRNA and gRNA.

Supplementary Figure 3. Sanger sequencing chromatograms of the GFP target sites.

Supplementary Figure 4. Schematic diagram of gene structures and gRNA target site

sequences of N. benthamiana PDS (a), RDR6 (b), and SGS3 (c) homoeologs.

Supplementary Figure 5. Lineage of CRISPR/Cas9-induced mutations in M1 progeny

derived from five representative M0 lines bi-allelic for both PDS homoeologs.


derived from two representative M0 lines heterozygous or chimeric for both PDS

homoeologs.

Supplementary Table 1. Sequences of tgtRNA fusion genes used in this study.

Supplementary Table 2. Identification of target mutations in both homoeologs of N.

benthamiana PDS, RDR6, and SGS3 genes by Sanger sequencing.

Supplementary Table 3. Summary of genotyping and phenotyping 30 regenerated M0 lines

Supplementary Table 4. Segregation of phenotypes and genotypes in M1 and M2 progeny

derived from two representative M0 lines heterozygous or chimeric for PDS-A and PDS-B.

Supplementary Table 5. Potential off-target sites of the gPDS-1 spacer.

Supplementary Table 6. List of primers used in plasmids construction, RT-PCR, cRT-PCR.

Supplementary Table 7. PCR primers and restriction enzymes used in mutation detection.

Supplementary Table 8. List of primers used for the analysis of off-target effects.

Supplementary Methods

Construction of SYNV vector for expression of Cas9 (pSYNV-Cas9)

To construct the pSYNV-Cas9 plasmid, we utilized the unique restriction sites Bsu36I and

NheI located in N and P genes in the pSYNV plasmid to facilitate sub-cloning. Fragment 1

encompassing partial N gene downstream of the Bsu36I site and the N/P J sequence (Bsu36I-

N/P J) was amplified from the pSYNV plasmid using the primer pair N-Bsu36I/F and Flag-

NPJ/R by Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd, Nanjing,

China). Fragment 2 containing the entire coding region of a human codon-optimized Cas9 gene,

as described by Feng et al37, was amplified from the pBGK01 plasmid38 with the primer pair

Cas9/F and Cas9/R. Fragment 3 containing the N/P J sequence and partial P gene sequence

upstream of the NheI site (N/P J-NheI) was amplified from the pSYNV plasmid using the primer

pair Cas9-NPJ/F and P-NheI/R. Each primer designed for these amplifications contains a 15-

to 20-nt overhang at its 5' end that is homologous to the 3' end of another PCR product, so that

the three PCR products can be ligated head-to-tail and cloned into the Bsu36I/NheI linearized

pSYNV plasmid to generate the pSYNV-Cas9 by using a ClonExpress MultiS One Step

Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing, China). Primer sequences used for

construction of this and all subsequent plasmids are listed in Supplementary Table 8.

Construction of SYNV vector for expression of gRNA and Cas9 (pSYNV-gRNA-Cas9)

The pSYNV-gRNA-Cas9 vector for expression of the gRNA containing the gGFP2 spacer

and Cas9 was generated similarly as described above. Briefly, the Bsu36I-N/P J fragment, the

gRNA sequence, and the N/P J-AhdI fragment containing the N/P J sequence and partial Cas9

sequence upstream of the AhdI site, were each amplified from the pSYNV, pBGK01, and

pSYNV-Cas9 plasmids by PCR using the three primer pairs, N-Bsu36I/F and GFP2-NPJ/R,

GFP2-gRNA/F and gRNA/R, and gRNA-NPJ/F and Cas9-AhdI/R, respectively. The resulting

three PCR products were inserted into the Bsu36I/AhdI linearized pSYNV plasmid by In-Fusion

Cloning.

Construction of SYNV vectors for expression of tgtRNA and Cas9 (pSYNV-tgtRNA-Cas9)

To construct SYNV-tgtRNA-Cas9 vectors carrying a single or tandem gRNAs flanked by

tRNAGly sequences, the tRNA-gRNA-tRNA and tRNA-gRNA-tRNA-gRNA-tRNA fusion

genes listed in Supplementary Table 1 were commercially synthesized by GenScript (Nanjing,

China). 15-nt overhangs derived from the 3'- (TTATTTGTCTAGGCC) and 5'- termini

(TAAACTACAGCCACA) of N/P J were added to the 5'- and 3'- ends of each of these fusion

genes during chemical synthesis to facilitate PCR amplification and subsequent In-Fusion

cloning. Next, these fusion genes were separately amplified by PCR using the primers NPJ-

tRNA/F and NPJ-tRNA/R. Also, the Bsu36I-N/P J and N/P J-AhdI fragments were amplified

from the pSYNV-Cas9 plasmid with the primer pairs N-Bsu36I/F and NPJ/R, and NPJ/F and

Cas9-AhdI/R, respectively. The three PCR fragments were cloned into the Bsu36I/AhdI-

linearized pSYNV-Cas9 vector by In-Fusion cloning.

Supplementary References

37 Feng, Z. et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell

Research 23, 1229-1232, doi:10.1038/cr.2013.114 (2013).

38 Fu, S. et al. Rice stripe virus interferes with s-acylation of remorin and induces its

autophagic degradation to facilitate virus infection. Molecular Plant 11, 269-287,

doi:10.1016/j.molp.2017.11.011 (2018).

39 Schwach, F. et al. An RNA-dependent RNA polymerase prevents meristem invasion

by potato virus X and is required for the activity but not the production of a systemic

silencing signal. Plant Physiol. 138, 1842-1852 (2005).

40 Li, F. et al. SGS3 Cooperates with RDR6 in triggering geminivirus-induced gene

silencing and in suppressing geminivirus infection in Nicotiana benthamiana. Viruses

9, pii: E247. doi: 10.3390/v9090247 (2017).

a

b

TAAACTACAGCCACAACTCTACCTCCCCCACTATGAATAAACGACCTAACATATAATATAAGAA

AAACCAACAGAAATCATAATATTTTATTTGTCTGTTTGTATTATTTGTCTAGGCCAACAAAGCAC

CAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTG

GTGCANNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG

TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGT

AGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATAAACTACAG

CCACAACTCTACCTCCCCCACTATGAATAAACGACCTAACATATAATATAAGAAAAACCAACAG

AAATCATAATATTTTATTTGTCTGTTTGTATTATTTGTCTAGGCCATG…(Cas9 ORF)…TAA

TAAACTACAGCCACAACTCTACCTCCCCCACTATGAATAAACGACCTAACATATAATATAAGAA

AAACCAACAGAAATCATAATATTTTATTTGTCTGTTTGTATTATTTGTCTAGGCCNNNNNNNNNN

NNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA

AAAAGTGGCACCGAGTCGGTGCTAAACTACAGCCACAACTCTACCTCCCCCACTATGAATAAA

CGACCTAACATATAATATAAGAAAAACCAACAGAAATCATAATATTTTATTTGTCTGTTTGTATTA

TTTGTCTAGGCCATG…(Cas9 ORF)…TAA

tgtRNA and Cas9 expression cassettes in SYNV-tgtRNA-Cas9

gRNA and Cas9 expression cassettes in SYNV-gRNA-Cas9

Supplementary Figure 1. Sequences of the gRNA and Cas9 expression cassettes in

the SYNV-tgtRNA-Cas9 (a) and SYNV-gRNA-Cas9 (b) vectors. The tgtRNA and Cas9

cassettes contain sequence elements in the order “N/P gene junction (N/P J) - tRNAGly -

gRNA spacer (denoted by 20 “N”) – gRNA scaffold - tRNAGly - N/P J - Cas9 ORF (only the

start and stop codons are shown)”. The organization of the gRNA and Cas9 cassette in the

SYNV-gRNA-Cas9 is similar but lacks the tRNA sequences flanking the gRNA. Within the

N/P J are the underlined adenine (A)-rich tracts that serve as templates for synthesis of

polyadenylated mRNA tails and termination of upstream mRNA transcription, the italicized

non-transcribed CC dinucleotides, and the boldfaced AAC start sites for initiation of

transcription of the adjacent downstream mRNA. Note that the sequences are shown in

antigenomic (mRNA) sense.

a b

mRNA transcription

RNase cleavage

gRNA

A(n)m7G5' UTR 3' UTRgRNAtRNA tRNA

Folding

96 nt50 nt 62 nt77 nt 77 nt

A(n)m7G

RNase Z RNase P

m7G A(n)

tRNA tRNA 3' UTR5' UTR

N/P J N/P J

A(n)m7G5' UTR 3' UTRgRNA

96 nt50 nt 62 nt

A(n)m7G

5' UTR- gRNA-3' UTR

RNase? RNase?

Cas9

N/P J N/P J

gRNA Cas9tRNA tRNA gRNA

Supplementary Figure 2. Schematic representation of transcription and processing

of the tgtRNA and gRNA. a, The primary tgtRNA transcript produced by SYNV-tgtRNA-

Cas9 (a) is processed precisely by RNase Z and RNase P to release the authentic gRNA

sequence. In contrast, the gRNA transcribed by SYNV-gRNA-Cas9 (b) is embedded in

virus-derived 5' and 3' untranslated regions (UTRs), which might be subjected to terminal

trimming off by cellular RNases non-specifically. Line color codes for viral UTRs, tRNA,

gRNA spacer, and scaffold are red, purple, blue, and black, respectively. N/P J, N/P gene

junction; m7G, 7-methylguanosine cap; A(n), polyadenylated tail.

SYNV-tgtRNA-Cas9 SYNV-gRNA-Cas9

gGFP1

d1

d3

d6

d7

d8

gGFP2

d2

d3

d4

d5

Supplementary Figure 3. Sanger sequencing chromatograms of the GFP target

sites. a, Schematic representation of the mGFP5 target sites in the N. benthamiana 16c

line. The red bars represent the gRNA target sites, with nucleotide coordinates indicated

on the top. The PAM sites are highlighted in red, and the restriction sites adjacent to the

Cas9 cleavage sites are underlined. b, Chromosomal DNA was extracted from upper

symptomatic leaves of plants systemically infected with SYNV vector expressing Cas9 and

gGFP1 (right panels) or gGFP2 (left panels), and DNA regions encompassing target sites

were amplified by PCR with specific primers and sequenced.

gGFP2 gGFP1

5'- CCTGTTCCATGGCCAACACTTGT…35…GATACCCAGATCATATGAAGCGG -3'

NcoI NdeIPAM PAM

mGFP5

a

b

1 792222 279

gPDS-1 gPDS-2 gPDS-3

5'-GCCGTTAATTTGAGAGTCCAAGG-83-TTGGTAGTAGCGACTCCATGGGG-175-CCGGAGCTAGACAATACAGTTAA- 3'

HinfI NcoI BfaI

PDS-A (Niben101Scf01283Ctg022)

PAM PAM PAM

PDS-B (Niben101Scf14708Ctg002)

gPDS-1 gPDS-2 gPDS-3

5'-GCCGTTAATTTGAGAGTCCAAGG-83-TTGGTAGTAGCGACTCCATGGGG-181-CCAGAGCTAGACAATACAGTTAA- 3'

HinfI NcoI BfaIPAM PAM PAM

gR6-1 gR6-4

5'-CCCCTCCTGACTCTTACCCAACT-1358-ATTCTCAGCTAACCAGCTGAGGG- 3'

Hpy188III PvuIIPAM PAM

RDR6-A (Niben101Scf12609Ctg016)

gR6-1 gR6-4

5'-CCCCTCCTGACTCTTACCCAACT-1356-ATTCTCAGCTAACCAGCTGAGGG- 3'

Hpy188III PvuIIPAM PAM

RDR6-B (Niben101Scf03832Ctg041)

gS3-1 gS3-2

5'-ACAAGAGTGGAAGCAGTGCTGGG-284-TGCCTCAACTGATCCCAAGGAGG- 3'

TscAI Eco130IPAM PAM

SGS3-A (Niben101Scf03392Ctg069)

gS3-1 gS3-2

5'-ACAAGAGTGGAAGCAGTGCTGGG-284-TGCCTCAACTGATCCCAAGGAGG- 3'

TscAI Eco130IPAM PAM

SGS3-B (Niben101Scf05468Ctg070)

a

b

c

Supplementary Figure 4. Schematic diagram of gene structures and gRNA target

site sequences of N. benthamiana PDS (a), RDR6 (b), and SGS3 (c) homoeologs.

Homoeologs are arbitrarily assigned to -A and -B for reference convenience, with gene IDs

shown in parentheses. Black rectangle, solid line, and red bar represent exon, intron, and

gRNA target site, respectively. The PAM sites are highlighted in red, and the restriction

sites overlapping with the Cas9 cleavage sites are underlined.


derived from five representative M0 lines bi-allelic for both PDS homoeologs. The

plant IDs of albino M1 progeny are labeled in blue letters. Dashes denote nucleotide

deletions, and boldface letters denote nucleotide insertions. The number of nucleotide

deletions (d#) or insertions (i#) are shown at the right side of each sequence, with in-frame

short deletions (d3, d6, and d9) highlighted in green letters.

22-1# A: GCCGTTAATTTGA----CCA d4

B: GCCGTTAATT-------CCA d7

22-2#/B: GCCGTTAATTTGA----CCA d4

22-5# GCCGTTAA---------CCA d9


GCCGTTAA---------CCA d9


B: GCCGTTAATTTGAGAGTTCCA i1

29-2# A: GCCGTTAATTT------CCA d6

B: GCCGTTAATTTGAG---CCA d3

GCCGTTAATTTGAGAGTTCCA i1

29-5# A: GCCGTTAATTT------CCA d6


18-1# A: GCCGTTAATTTGAGA-TCCA d1

GCCGTTAAT--------CCA d8

B: GCCGTTAATTTGAG----CA d4

18-2#/A: GCCGTTAATTTGAGA-TCCA d1

18-4# GCCGTTAAT--------CCA d8

B: GTCGTTAATTTGAG----CA d4

GCCGTTAATTT------CCA d6


B: GCCGTTAATTTG-----CCA d5

8-3#/ A: GCCGTTAATTTGAGAGTTCCA i1

8-4# B: GCCGTTAATTTGAGAGT--- d3

M0-8 A: GCCGTTAATTTGAGA-TCCA d1


B: GCCGTTAATTTGAGAGT--- d3

GCCGTTAATTTG-----CCA d5

M0-18 A: GCCGTTAATTTGAGA—TCCA d1

GCCGTTAAT--------CCA d8



M0-22 A: GCCGTTAATTTGA----CCA d4




M0-29 A: GCCGTTAATTTGA----CCA d4




8-2#/ A: GCCGTTAATTTGAGA-TCCA d1

8-5# GCCGTTAATTTGAGAGTTCCA i1

B: GCCGTTAATTTGAGAGT--- d3

GCCGTTAATTTG-----CCA d5


B: GCCGTTAATTT------CCA d6

18-5# A: GCCGTTAAT--------CCA d8







B: GCCGTTAA---------CCA d9








M0 No. Genotypes M1 ID Genotypes

M0-42 A: GCCGTTAATTTGAG---CCA d3

GCCGTTAATTT-----TCCA d5

B: GCCGTTAATTT-----TCCA d5


M1 ID Genotypes

42-1# A: GCCGTTAATTT-----TCCA d5


42-2# A: GCCGTTAATTTGAG---CCA d3

GCCGTTAATTT-----TCCA d5


42-3#/A: GCCGTTAATTTGAG---CCA d3

42-5# GCCGTTAATTT-----TCCA d5


42-4# A: GCCGTTAATTT-----TCCA d5



derived from two representative M0 lines heterozygous or chimeric for both PDS

homoeologs. Dashes denote nucleotide deletions, and boldface letters denote nucleotide

insertions. The number of nucleotide deletions (d#) or insertions (i#) are shown at the right

side of each sequence, with the in-frame short deletions (d3 and d6) highlighted in green

letters.

M0-34 A: GCCGTTAATTTGAGAGTCCA WT

GCCGTTAATTTGAGA-TCCA d1

GCCGTTAATTTGAGA--CCA d2


GCCGTTAATT-------CCA d7


B: GCCGTTAATTTGAGAGTCCA WT

GCCGTTAATTTGAG---CCA d3

GCCGTTAATTTGA----CCA d4

34-1#/A: GCCGTTAATTTGAGAGTCCA WT

34-4# B: GCCGTTAATTTGAGAGTCCA WT

34-3# A: GCCGTTAATTTGAGAGTTCCA i1


34-2# A: GCCGTTAATTTGAGAGTCCA WT



M0 No. Genotypes M1 ID Genotypes

M0-7 A: GCCGTTAATTTGAGAGTCCA WT



GCCGTTAA------AGTCCA d6

M1 ID Genotypes

7-1# A: GCCGTTAATTTGAGAGTCCA WT





GCCGTTAA------AGTCCA d6

7-3#/ A: GCCGTTAATTTGAGAGTCCA WT

7-4# GCCGTTAATTTGA----CCA d4

B: GCCGTTAA------AGTCCA d6

Supplementary Table 1. Sequences of tgtRNA fusion genes used in this study.

Gene and

architecture Sequence (5'→3')

tRNA-

gGFP1-

tRNA

AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACC

CGGGTTCGATTCCCGGCTGGTGCAGATACCCAGATCATATGAAGGTTTTAGAG

CTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG

CACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTG

CCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCA

tRNA-

gGFP2-

tRNA


CGGGTTCGATTCCCGGCTGGTGCAACAAGTGTTGGCCATGGAACGTTTTAGAG




tRNA-

gPDS-1-

tRNA


CGGGTTCGATTCCCGGCTGGTGCAGCCGTTAATTTGAGAGTCCAGTTTTAGAG




tRNA-

gPDS-2-

tRNA


CGGGTTCGATTCCCGGCTGGTGCATTGGTAGTAGCGACTCCATGGTTTTAGAG




tRNA-

gPDS-3-

tRNA


CGGGTTCGATTCCCGGCTGGTGCATTAACTGTATTGTCTAGCTCGTTTTAGAG




tRNA-

gR6-1-

tRNA


CGGGTTCGATTCCCGGCTGGTGCAAGTTGGGTAAGAGTCAGGAGGTTTTAGAG




tRNA-

gR6-4-

tRNA


CGGGTTCGATTCCCGGCTGGTGCAATTCTCAGCTAACCAGCTGAGTTTTAGAG




tRNA-

gS3-1-

tRNA


CGGGTTCGATTCCCGGCTGGTGCAACAAGAGTGGAAGCAGTGCTGTTTTAGAG




Supplementary Table 1. (Continued) Sequences of tgtRNA fusion genes used in this study.

tRNA-

gS3-2-

tRNA


CGGGTTCGATTCCCGGCTGGTGCATGCCTCAACTGATCCCAAGGGTTTTAGAG




tRNA-

gR6-1-

tRNA-

gS3-1-

tRNA


CGGGTTCGATTCCCGGCTGGTGCAAGTTGGGTAAGAGTCAGGAGGTTTTAGAG



CCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAACAAGAGTGGAAGCA

GTGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA

ACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGT

AGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCA

tRNA-

gR6-4-

tRNA-

gS3-2-

tRNA


CGGGTTCGATTCCCGGCTGGTGCAATTCTCAGCTAACCAGCTGAGTTTTAGAG



CCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATGCCTCAACTGATCC

CAAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA



tRNA-

gPDS1-

tRNA-

gPDS3-

tRNA


CGGGTTCGATTCCCGGCTGGTGCAGCCGTTAATTTGAGAGTCCAGTTTTAGAG



CCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATTAACTGTATTGTCT

AGCTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA



The sequences are annotated as follows:

pre-tRNA: magenta background.

gRNA scaffold: white background.

gRNA spacer: white background with blue letters.

Supplementary Table 2. Identification of target mutations in both homoeologs of N.

benthamiana PDS, RDR6, and SGS3 genes by Sanger sequencing.

gRNA Target

gene Sequences

Mutation

type Count

Mutation

frequency

gPDS-1

PDS-A

GCCGTTAATTTGAGAGTCCAAGG

GCCGTTAATTTGAGAG-CCAAGG

GCCGTTAATTTGAGA--CCAAGG

GCCGTTAATT-------CCAAGG

GCCGT------------CCAAGG

GCCGTTAATTTGAGAGTTCCAAGG

WT

d1

d2

d7

d12

i1

2

2

1

3

1

1

8/10

PDS-B

GCCGTTAATTTGAGAGTCCAAGG

GCCGTTAATTTGAGAG-CCAAGG

GCCGTTAATTTGAG--TCCAAGG

GCCGTTAATTTG----TCCAAGG

GCCGTTAAT------CTCCAAGG

GCCGTTAATTTGAGAGTTCCAAGG

WT

d1

d2

d4

d6

i1

3

1

1

1

1

3

7/10

gR6-4

RDR6-A

ATTCTCAGCTAACCAGCTGAGGG

ATTCTCAGCTAACCA--TGAGGG

ATTCTCAGCTAAC----TGAGGG

ATTCTCAGCTAA-----TGAGGG

ATTCTCAGCT-----GGTGAGGG

ATTCTCAGC--------TGAGGG

WT

d2

d4

d5

d5s1

d8

4

1

1

2

1

1

6/10

RDR6-B


ATTCTCAGCTAACCA---GAGGG

ATTCTCAGCTA-----CTGAGGG

ATTCTCAGCT-------TGAGGG

ATTCTCAGCTAAT---------G

ATTCTCAGCTAACCAGCTTGAGGG

WT

d3

d5

d7

d9

i1

3

1

2

1

2

1

7/10

gS3-2

SGS3-A

TGCCTCAACTGATCCCAAGGAGG

TGCCTCAACTGATCCC-AGGAGG

TGCCTCAACTGATCC--AGGAGG

TGCCTCAACTG------AGGAGG

WT

d1

d2

d6

2

4

2

2

8/10

SGS3-B



TGCCTCAACTGATCC---GGACC

TGCCTCAACTGAT------GAGG

TGCCTCA----------AGGAGG

TGCCTCAACTGATCCCAAAGGAGG

WT

d1

d3

d6

d10

i1

4

2

1

1

1

1

6/10

Supplementary Table 2. (Continued) Identification of target mutations in both homoeologs of

N. benthamiana PDS, RDR6, and SGS3 genes by Sanger sequencing.

Deleted nucleotides are denoted by dash symbols, and inserted nucleotides or substitutions are

shown in boldface and italicized letters, respectively. Mutation type: WT, wild-type sequence

with no mutation detected; d#, number of bases deleted from target site; i#, number of bases

inserted at target site; i#a, the same number of insertion as in i# but different nucleotide was

inserted; s#, number of bases substitution at target site.

gR6-4

/gS3-2

RDR6-A



ATTCTCAGCTAACC---TGAGGG

ATTCTCAGCTGGTT---TGAGGG

ATTCTCAGCTAA-----TGAGGG

ATTCTCAGC--------TGAGGG

WT

d2

d3

d3s4

d5

d8

5

1

1

1

1

1

5/10

RDR6-B



ATTCTCAGCTAACC---TGAGGG

ATTCTCAGCT-------TGAGGG

ATTCTCAGCTA------------

WT

d2

d3

d7

d12

5

1

2

1

1

5/10

gR6-4

/gS3-2

SGS3-A




TGCCTCAACTGATCCCATAGGAGG

WT

d1

i1

i1a

3

4

2

1

7/10

SGS3-B



TGCCTCAACTGATCC---GGACC

TGCCTCAACTGAT----AGGAGG

TGCCTCAA---------AGGAGG


WT

d1

d3

d4

d9

i1

2

2

1

1

1

3

8/10

Supplementary Table 3. Summary of genotyping and phenotyping 30 regenerated M0 lines

WT, wild-type sequence without mutation; d# and i#, # of bp deleted and inserted from the

target site; Green letters indicates in-frame deletions. The zygosity of homozygote (Ho), bi-

allelic (Bi), heterozygote (He), and chimera (Ch) in M0 plants is putative. NT, not tested.

Line

No. Phenotype

Genotype

Virus HinfI

susceptible? Percentage

Zygosity

PDS-A/-B

Mutation type

PDS-A/-B Genotype

M0-1

Albino

No

17/30

(56.7%)

Bi/Bi d5d7/d5d1 aabb +

M0-2 No Bi/Bi d1d7/d4d7 aabb +

M0-4 No Bi/Bi d1d7/d1i1 aabb +

M0-5 No Ho/Ho i1i1/i1i1 aabb +

M0-9 No Bi/Ho i1d2/i1i1 aabb +

M0-11 No Bi/Bi d2d8/i1d8 aabb +

M0-8

Normal

No Bi/Bi d1i1/d3d5 aabb +





M0-29 No Bi/Bi d4d6/d3i1 aabb +

M0-31 No Bi/Bi d7i1/d7d9 aabb +


M0-33 No Bi/Bi d7i1/d7d9 aabb +


M0-42 No Bi/Bi d3d5/d5d9 aabb -

M0-7 Partial

11/30

(36.6%)

He/He WTd4/WTd6 AaBb -

M0-34 Partial Ch/Ch

WT,d1,d2,d6,

d7,i1/WT,d3,d

4

Chimera +

M0-10 Partial NT NT NT +









M0-3 Yes 2/30 (6.7%)

WT/WT WT/WT AABB -

M0-6 Yes WT/WT WT/WT AABB +

Supplementary Table 4. Segregation of phenotypes and genotypes in M1 and M2 progeny

derived from two representative M0 lines heterozygous or chimeric for PDS-A and PDS-B.

M0

No.

Genotype

PDS-A/-B

M1 progeny M2 progeny

phenotype ID Phenotype

Genotype

PDS-A/-B

Zygosity

PDS-A/-B Normal Albino

7 WTd4/

WTd6

7-1# Normal WTd4/WTWT He/WT 89 0

7-2# Normal d4d4/WTd6 Ho/He 105 0

7-3# Normal WTd4/d6d6 He/Ho 124 0

7-4# Normal WTd4/d6d6 He/Ho 91 0

34

WT,d1,d2

d6,d7,i1/

WT,d3,d4

34-1# Normal WTWT/WTWT WT/WT 97 0

34-2# Normal WTi1/WTWT He/WT 157 0

34-3# Normal i1i1/WTWT Ho/WT 144 0

34-4# Normal WTWT/WTWT WT/WT 128 0

WT, wild-type sequence with no mutation detected; d# and i# denote # of bp deleted and

inserted from the target site, and the in-frame deletion types (d3 and d6) are highlighted in green

letters. He, heterozygote; Ho, homozygote.

Supplementary Table 5. Potential off-target sites of the gPDS-1 spacer.

Name Locus Sequence No. of

mismatches

HinfI

site

PDS-A

PDS-B

Niben101Scf01283

Niben101Scf14708 GCCGTTAATTTGAGAGTCCAAGG 0 Yes

OT1 Niben101Scf01169 GCCGaaAATTTGAGAGTCCACGG 2 Yes

OT2 Niben101Scf00953 GCCGTTAAgTTGAaAtTCCATGG 3 No

OT3 Niben101Scf04197 GCCGTTAATTTtAtAGTCaATGG 3 No

OT4 Niben101Scf02210 ataGTgAATTTGAGAGTCCATGG 4 Yes

OT5 Niben101Scf00693 aCCccgAATTTGAGAGTCCAGGG 4 Yes

OT6 Niben101Scf03072 tgCGTgAgTTTGAGAGTCCAGGG 4 Yes

OT7 Niben101Scf03141 GCaaTgAgTTTGAGAGTCCAGGG 4 Yes

OT8 Niben101Scf01023 GgtGTTAATTTGAGAGTCatTGG 4 Yes

OT9 Niben101Scf00698 GCCGTTAATTTaAGAGTCtgATG 3 Yes

OT10 Niben101Scf02214 ttgGTTAATTTGAGAGTCtATTT 4 Yes

OT11 Niben101Scf07398 aCttgaAATTTGAGAGTCCATAT 5 Yes

OT12 Niben101Scf04016 tgCtTTAATTTGAGAGTCggAAT 5 Yes

OT13 Niben101Scf02793 atCaaaAATTTGAGAGTCCAGAA 5 Yes

The PAM site (NGG) is marked in red, the HinfI site underline, and the mismatched nucleotides

shown in lower-case letters.

Supplementary Table 6. List of primers used in plasmids construction, RT-PCR, cRT-PCR.

Primer Sequence (5' → 3') Application

N-Bsu36I/F AGATCTTCAATGTCCTCAGGTTC Construction of pSYNV-Cas9

Flag-NPJ/R gtggtccttatagtccatGGCCTAGACAAATAATACAAACAG Construction of pSYNV-Cas9

Cas9/F ATGGACTATAAGGACCACGAC Construction of pSYNV-Cas9

Cas9/R TTACTTTTTCTTTTTTGCCTGGC Construction of pSYNV-Cas9

Cas9-NPJ/F gcaaaaaagaaaaagtaaTAAACTACAGCCACAACTCTACC Construction of pSYNV-Cas9

P-NheI/R TTTTCCGTATGACATGCTAGCC Construction of pSYNV-Cas9

GFP2-NPJ/R gttccatggccaacacttgtGGCCTAGACAAATAATACAAACAG Construction of pSYNV-gRNA-Cas9

GFP2-gRNA/F acaagtgttggccatggaacGTTTTAGAGCTAGAAATAGCAAG Construction of pSYNV-gRNA-Cas9

gRNA/R GCACCGACTCGGTGCCACTTTTTC Construction of pSYNV-gRNA-Cas9

gRNA-NPJ/F aagtggcaccgagtcggtgcTAAACTACAGCCACAACTCTAC Construction of pSYNV-gRNA-Cas9

Cas9-AhdI/R TTGTCTTGCCGGACTGCTT Construction of pSYNV-gRNA-Cas9

NPJ/R GGCCTAGACAAATAATACAAACAG Construction of pSYNV-tgtRNA-Cas9

NPJ-tRNA/F ttatttgtctaggccAACAAAGCACCAGTGGTCTAG Construction of pSYNV-tgtRNA-Cas9

NPJ-tRNA/R tgtggctgtagtttaTGCACCAGCCGGGAATC Construction of pSYNV-tgtRNA-Cas9

NPJ/F TAAACTACAGCCACAACTCTACCT Construction of pSYNV-tgtRNA-Cas9

GFP-1/F GATACCCAGATCATATGAAG RT-PCR detection of gRNA insert

gRNA/R GCACCGACTCGGTGCCACTTTTTC RT-PCR detection of gRNA insert

Cas9 /F ATGGACTATAAGGACCACGAC RT-PCR detection of Cas9 insert

Cas9-AhdI/R TTGTCTTGCCGGACTGCTT RT-PCR detection of Cas9 insert

SYNV-M/F ATGGCAGGTATATACGCAGTTTCAA RT-PCR detection of SYNV infection

SYNV-M/R TCAGTCTCATCTTCAAAGTATGTAGGA RT-PCR detection of SYNV infection

NbActin/F CAATCCAGACACTGTACTTTCTCTC RT-PCR detection of Actin mRNA

NbActin/R AAGCTGCAGGTATCCATGAGACTA RT-PCR detection of Actin mRNA

Scaffold/F GTTTTAGAGCTAGAAATAGCAAG cRT-PCR mapping of gRNA termini

GFP-2/R GTTCCATGGCCAACACTTGT cRT-PCR mapping of gRNA termini

The restriction sites are underlined.

Sequences in lowercase letters are designed to facilitate In-Fusion cloning.

Supplementary Table 7. PCR primers and restriction enzymes used in mutation detection.

Target loci Oligo sequence (5' → 3')

For: forward; Rev: Reverse

PCR product

(bp)*

Restriction

site

Digestion

products (bp)

GFP For: ATGAAGACTAATCTTTTTCTCTTTCTCATC

Rev: TTAAAGCTCATCATGTTTGTATAGTTCATC 792

NdeI

NcoI

499, 293

563, 229

PDS-1 For: AATCTTTTTTACTTTTGCATATAAATTTGTG

Rev: AACTTATGCCCCATGCAGTC 329/328 HinfI 206/205, 123

PDS-2 For: TTAGGTTCACAAGTGGGACAATCTTC

Rev: CAGCATCACACTTTCGCATTCAAAAC 333 NcoI 227, 106

PDS-3 For: TGTTTTGAATGCGAAAGTGTGATGCTG

Rev: AACTAATAGAATGATCTTCCTTCCAAAGAAAG 291/285 BfaI 175, 116/110

PDS-1&3 For: TTAGGTTCACAAGTGGGACAATCTTC

Rev: GTTGGGCGTGAGGAAGTACG 492/486 n/a n/a

RDR6-1 For: CAAGTATGGAGATGTAGATTAAAG

Rev: TTTGCGGACTCTGAAGATGC 152 Hpy188III 112, 40

RDR6-4 For: ATTGTAGAGGTTAGGAGATTGGTTAT

Rev: GCATAAGTTGATGAAAAGCACTGTCC 461/460 PvuII 318/317, 143

SGS3-1 For: GGTGTTGGAGATATGGGCTTTAACTCTG

Rev: GACCAATCCCATCCATTCTTCAGAGCAG 368 TscAI 278, 90

SGS3-2 For: GGTGTTGGAGATATGGGCTTTAACTCTG

Rev: GCGTCTCATAACTCATTTCAGCCACATC 547 Eco130I 392, 155

PDS-1A For: AACACAAACAGTTAAGTACTTCTTTAATCG

Rev: GTGGCACTTGGAGTACGAATCCTT 783 HinfI -

PDS-1B For: GAAAGATTCAACAATTAGTATTTCTTTAAGCC

Rev: ATGGCACTGGGAGTACGAATTCTA 797 HinfI -

RDR6-4A For: CAAATTCGAATCCACAGAGAACTGCA

Rev: CAGAAGGAGGGTTGACACTTAAATG 408 PvuII -

RDR6-4B For: CAAATTCAAATCCCCAGAGAACTGCG

Rev: ATAAGCAGAAACAGGGTTGACACTTAAAATC 414 PvuII -

SGS3-2A For: CAGCTATATGATGGAGGTCTTGG

Rev: GATTCGAAAAGTTCTTTGATCCAACG 302 Eco130I -

SGS3-2B For: CAGCTATATGACGGAGGTTCTGT

Rev: GAGATTCGAAAAGTTCTTTGAACCAACA 304 Eco130I -

*The sizes of PCR products and digestion products for A and B homoeologs, if differed, are

denoted by #/#. n/a: not applicable.

Supplementary Table 8. List of primers used for the analysis of off-target effects.

Site

name

Forward primer sequence (5' → 3') Reverse primer sequence (5' to 3') Size

(bp)

PDS-1A

OT-1

OT-2

OT-3

OT-4

OT-5

OT-6

OT-7

OT-8

OT-9

OT-10

OT-11

OT-12

OT-13

CAGAATATTTGAAAAAGATAAGGAATTTTG

ATTTTTGGTATTTTTTGGGCTTTTAG

CAATGCCCTTATTTCATTAATCTTGT

CCACCGATCCAGTAAAGCTGATAC

CAAGTTCAATTTGCAGACTCATA

TTCTCATCTCTATGTCTGATTCG

GTATTTGCAAGTTTGACTCTAAG

GTTAATCCTCGTTATGTTCCCTTGCTCT

TTAGCATTGAATGGGTCCAGG

ATTTTCAATTATGCTTGTTTATTCC

TCTTTGCCAGGATGTAATTAAGA

GCAACATGAAGTTAGTTTGAGCA

TCCTGTAATTTTCTTTGTGATTCG

TGTGAAAAGAAACTAGCTAACTGGAA

TAACAAATTCCTTTGCAAGCAAA

ATTCCGAGCCGGGTCC

TTAGTCATTCCCGAGTTCTGTGGTTT

CCACCATTCAAATAGGCAATAACA

GACCCTAGGATTTGGAGGTT

TACAAATCCAATTTGTAGACTCAC

CCTAATATATAAGCATACTAGTATCAAAC

CGAAATCAATCAATTAGTTGGTAAAGAT

GCCTCCAACCCACCATAAA

CCAATTCCAAGCTTTGTAACC

GCACATAGGAGTAATAGTGGGTGA

GGGGCAGAAGGTAATTAGCAG

TTCCCCTTTCTTGAGTTCCA

CTCCATATGGCCCTCTTACAG

245

89

390

307

114

141

154

552

417

600

586

596

699

402

highly efficient dna-free plant genome editing using ...10.1038...lineage of crispr/cas9-induced...

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