Titel:
CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum
Journal:
Plant Molecular Biology
Authors:
Junping Gao1†, Genhong Wang1†, Sanyuan Ma1, Xiaodong Xie2, Xiangwei Wu1,
Xingtan Zhang2, Yuqian Wu2, Ping Zhao1, Qingyou Xia1*
Author affiliation:
1
State Key Laboratory of Silkworm Genome Biology, Southwest University,
Chongqing 400715, China
2
School of Life Science, Chongqing University, Chongqing 400030, China
†
These authors contributed equally to this work.
*
To whom correspondence should be addressed:
Email: xiaqy@swu.edu.cn
Online Resource 1 List of primers used in this study.
Primer name
Sequence (5'-->3')
U6-26-F
CCGGAATTCAGCTTTCGTTTTCTTCTTTTTAACT
U6-26-R
ATTTGCGGCCGCCAGAAAACGAAGAGAAAAACCC
PDS-1-R
TGGACTCTCAAATTAACGGCAATCACTACTTCGACTCT
PDS-1-F
GCCGTTAATTTGAGAGTCCAGTTTTAGAGCTAGAAATAGC
PDS-2-R
GAGATTGTTATTGCTGGTGCAATCACTACTTCGACTCT
PDS-2-F
GCACCAGCAATAACAATCTCGTTTTAGAGCTAGAAATAGC
PDS-3-R
CCTAGGACATCTCTTGCCTCAATCACTACTTCGACTCT
PDS-3-F
GAGGCAAGAGATGTCCTAGGGTTTTAGAGCTAGAAATAGC
PDS-4-R
TCATCATCTTTCCATGCAGCAATCACTACTTCGACTCT
PDS-4-F
GCTGCATGGAAAGATGATGAGTTTTAGAGCTAGAAATAGC
PDR6-1-R
ACTCTAGCTTCTGCATCTACAATCACTACTTCGACTCT
PDR6-1-F
GTAGATGCAGAAGCTAGAGTGTTTTAGAGCTAGAAATAGC
PDR6-2-R
TCCTGGTTTGATGATTCCACAATCACTACTTCGACTCT
PDR2-2-F
GTGGAATCATCAAACCAGGAGTTTTAGAGCTAGAAATAGC
PDS1-F
GCTTTACTTGAGAAAAGCTCTC
PDS1-R
ACCTTTAAAGGATTAAAGTCCTT
E2S
TAAAGGAGATGCACCAACTTGT
E2A
AAGTATATCTTGCCATTCAGGTAGT
PDS3-F
ATGGAGGGTGGTGAAGACAG
PDS3-R
CGAAGTGTTGGTTGAATTAAGTAA
E4S
TTAATTGTCGAGAAAATAGTACAGC
E4A
TAAAGGAGATGCACCAACTTGT
PDR6-1-F
ATTTTCATTAGAACACTCTCCGA
PDR6-1-R
TTACACAGAAAGTAAAATGGAGATA
PDR6-2-F
TACATAGGATTTTTCTTGTACATCA
PDR6-2-R
TATATAAGCAGATGATCTTTGTGGT
Offtarget-F CTCTAGCCTTGATCTTATCATAGC
Offtarget-R TTAGGCTACTGCTGAACATCTC
Usage
PCR the U6-26 gRNA
chimera
For constructing gRNA of
NtPDS target 1
For constructing gRNA of
NtPDS target 2
For constructing gRNA of
NtPDS target 3
For constructing gRNA of
NtPDS target 4
For constructing gRNA of
NtPDR6 target 1
For constructing gRNA of
NtPDR6 target 2
PCR NtPDS target 1 region
PCR NtPDS target 2 region
PCR NtPDS target 3 region
PCR NtPDS target 4 region
PCR NtPDR6 target 1
region
PCR NtPDR6 target 2
region
PCR potential off-target
site region
Online Resource 2 Targeted mutagenesis on the NtPDR6 gRNA2 target site. The indel rate was
calculated based on the ratio of mutated clonal amplicons vs. total randomly sequenced clonal
amplicons. The target sequences of the gRNA are highlighted with green. The indels are shown in red
letters and dashes. PAM, the protospacer adjacent motif.
Online Resource 3 Targeted mutagenesis on the NtPDS gRNA4 target site. The indel rate was
calculated based on the ratio of mutated clonal amplicons vs. total randomly sequenced clonal
amplicons. The target sequences of the gRNA are highlighted with green. The indels are shown in red
letters and dashes. PAM, the protospacer adjacent motif.
Online Resource 4 Targeted mutagenesis on gRNA2 and gRNA4 target site of NtPDS. a, b PCR
products from the genomic DNA of tobacco protoplasts transfected with the one expression vector, that
contained the gRNA2 and gRNA4 of NtPDS, were sequenced and aligned to the reference genome
sequence. The indel rate was calculated based on the ratio of mutated clonal amplicons vs. total
randomly sequenced clonal amplicons. The target sequences of gRNA are highlighted with green. The
indels are shown in red letters and dashes. PAM, the protospacer adjacent motif.
Online Resource 5 Off-target analysis of the Cas9/gRNA-induced mutations. a One potential
off-target site for PDR6-gRNA2 was examined. b PCR/RE assay to detect the potential mutations. The
PCR products were amplified from the genomic DNA of the transgenic plant 14 of the PDR6 gRNA2
target and digested with BstNI.
Online Resource 6 Phenotype of PDR6 mutation in T0 transgenic plant of tobacco plants. a Branched
phenotypes comparison between WT and transgenic plant. The plants were transplanted in soil for 1
week. The left one is a T0 plant with nontransgenic wild-type tobacco, while the right one is a
transgenic plant with PDR6 mutated. b Detail of branched phenotypes of pdr6 mutant. The scale bars
are 3 cm (a).
AGCTTTCGTTTTCTTCTTTTTAACTTTCCATTCGGAGTTTTTGTATCTTGTTTCATAGTTTGTCCCAGGATTAGAATGAT
TAGGCATCGAACCTTCAAGAATTTGATTGAATAAAACATCTTCATTCTTAAGATATGAAGATAATCTTCAAAAGGCCC
CTGGGAATCTGAAAGAAGAGAAGCAGGCCCATTTATATGGGAAAGAACAATAGTATTTCTTATATAGGCCCATTTAAG
TTGAAAACAATCTTCAAAAGTCCCACATCGCTTAGATAAGAAAACGAAGCTGAGTTTATATACAGCTAGAGTCGAAG
TAGTGATTNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA
ACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCAAAATTTTCCAGATCGATTTCTTCTTCCTCTGTTCTTCGGC
GTTCAATTTCTGGGTTTTTCTCTTCGTTTTCTG
Online Resource 7 The sequence of the gRNA expression vector. The Arabidopsis thaliana U6-26
promoter is shown in green, and the gRNA scaffold in blue. The target sequence is in red.
cctgcaggtcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcct
cggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagt
ggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgataacatggtggagcacgacacacttgtctact
ccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagata
gtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtg
gaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagtt
catttcatttggagaggacctcgacctcaacacaacatatacaaaacaaacgaatctcaagcaatcaagcattctacttctattgcagcaatttaaatcatttcttttaaagcaaaagcaattttctga
aaattttcaccatttacgaacgatactcgagggggggtaccccccaatacttgttgtggactagtcgagctcgATGGATTACAAGGATCACGATGGAGATTACA
AAGATCACGATATAGATTACAAAGATGATGATGATAAAATGGCACCAAAAAAGAAGAGAAAGGTTGGAATACAT
GGTGTGCCAGCTGCAGATAAGAAGTACTCTATAGGACTTGATATAGGTACAAATTCAGTTGGATGGGCTGTGATTA
CTGATGAATATAAGGTTCCTTCTAAGAAATTCAAGGTGTTGGGTAATACTGATAGGCACTCAATCAAGAAAAACCT
TATTGGTGCTCTTTTGTTTGATAGTGGTGAAACTGCTGAGGCAACAAGACTTAAAAGGACTGCAAGAAGGAGATA
CACAAGGAGAAAGAACAGAATATGTTATTTGCAAGAAATATTTTCTAATGAGATGGCTAAGGTTGATGATTCTTTC
TTTCATAGGCTCGAAGAGTCATTTCTTGTGGAAGAGGATAAGAAACATGAAAGACACCCAATCTTCGGAAATATT
GTTGATGAAGTGGCTTACCATGAGAAATATCCTACAATCTATCACTTGAGAAAGAAATTAGTTGATTCTACTGATA
AGGCAGATCTTAGGCTCATATACCTTGCTTTGGCACATATGATCAAGTTTAGAGGACACTTCTTGATTGAAGGAGA
TCTTAATCCAGATAACTCAGATGTTGATAAACTCTTTATTCAACTTGTGCAGACATATAACCAACTTTTCGAAGAG
AATCCAATTAATGCTAGTGGAGTTGATGCTAAAGCAATCTTGTCCGCAAGACTCTCTAAGTCTAGAAGATTGGAAA
ATCTCATCGCTCAGTTGCCAGGAGAAAAGAAGAATGGTCTCTTTGGTAACCTTATTGCATTATCTCTCGGTCTTACA
CCTAATTTTAAGTCAAACTTCGATCTTGCTGAAGATGCAAAATTGCAACTCTCTAAGGATACTTACGATGATGATT
TGGATAATCTTCTCGCTCAGATTGGAGATCAATATGCAGATCTCTTTCTTGCTGCAAAAAACTTGAGTGATGCTAT
ACTTTTGTCCGATATCCTTAGGGTTAACACTGAAATCACAAAGGCTCCACTTTCTGCATCAATGATAAAGAGATAC
GATGAGCATCACCAAGATTTGACTTTACTCAAGGCACTCGTTAGACAACAGCTTCCTGAAAAGTATAAGGAGATTT
TCTTTGATCAGTCTAAGAATGGTTACGCTGGTTACATAGATGGAGGTGCATCTCAAGAAGAGTTCTATAAGTTCAT
TAAACCAATCTTGGAAAAGATGGATGGAACAGAAGAGCTTTTGGTTAAACTTAACAGAGAAGATCTTCTCAGAAA
GCAGAGGACTTTTGATAATGGTTCAATCCCACATCAAATCCACCTTGGTGAATTGCATGCTATTTTGAGGAGACAA
GAGGATTTTTACCCTTTCCTTAAGGATAACAGAGAAAAGATCGAGAAGATATTGACTTTTAGAATACCATACTACG
TTGGACCTCTTGCTAGAGGTAATAGTAGGTTCGCATGGATGACTAGGAAATCCGAAGAGACTATAACACCATGGA
ATTTTGAAGAGGTTGTGGATAAGGGTGCTAGTGCACAGTCCTTTATTGAAAGAATGACAAACTTCGATAAGAATTT
GCCAAACGAGAAGGTTCTCCCTAAGCATAGTCTTTTGTACGAATACTTCACTGTGTACAATGAGCTTACAAAGGTT
AAATATGTGACTGAAGGAATGAGGAAGCCTGCTTTTCTTTCAGGAGAGCAGAAGAAAGCAATTGTTGATCTTCTCT
TCAAAACTAACAGAAAGGTTACAGTGAAGCAACTTAAGGAAGATTACTTCAAGAAAATAGAGTGCTTCGATTCTG
TTGAAATCTCAGGAGTGGAGGATAGATTCAATGCTTCTTTGGGTACTTATCATGATCTTTTGAAGATTATTAAGGA
TAAAGATTTTCTCGATAATGAGGAGAACGAAGATATTCTTGAGGATATAGTTTTGACTCTCACACTCTTCGAAGAT
AGAGAGATGATTGAAGAGAGGCTCAAAACATACGCTCACCTTTTCGATGATAAGGTTATGAAACAACTTAAGAGG
AGAAGGTATACTGGATGGGGTAGATTGTCAAGAAAACTTATTAATGGTATCAGGGATAAGCAGTCTGGAAAGACT
ATTCTCGATTTCCTCAAGTCAGATGGATTCGCTAACAGAAACTTCATGCAATTGATCCATGATGATTCTCTCACTTT
TAAAGAAGATATCCAAAAGGCTCAGGTTTCTGGACAGGGAGATTCTCTTCATGAGCACATTGCTAATCTTGCAGGA
TCACCAGCAATCAAGAAAGGTATATTGCAAACTGTTAAGGTTGTGGATGAATTAGTTAAAGTGATGGGTAGACAT
AAGCCTGAGAATATCGTGATTGAAATGGCTAGGGAGAACCAAACTACACAGAAAGGTCAAAAGAATAGTAGAGA
AAGGATGAAAAGGATAGAAGAGGGAATCAAGGAGTTGGGTTCCCAAATACTCAAGGAACATCCTGTTGAGAACA
CACAATTGCAGAACGAAAAACTTTACTTGTACTACCTTCAGAACGGAAGAGATATGTATGTGGATCAAGAACTTG
ATATTAATAGGCTTAGTGATTATGATGTTGATCACATAGTGCCACAATCTTTTCTTAAGGATGATTCAATCGATAAT
AAGGTTCTTACTAGATCTGATAAGAATAGGGGAAAGTCTGATAACGTGCCTTCCGAAGAGGTTGTTAAGAAGATG
AAGAACTACTGGAGACAGTTACTCAACGCTAAACTCATCACACAAAGAAAGTTCGATAATCTTACTAAAGCTGAA
AGGGGAGGTCTTTCAGAGTTGGATAAAGCAGGTTTTATTAAGAGACAGTTGGTTGAAACTAGGCAAATTACAAAA
CATGTGGCTCAAATCCTTGATAGTAGGATGAATACAAAGTACGATGAAAACGATAAATTGATCAGAGAGGTTAAG
GTTATTACTCTCAAGAGTAAACTTGTTTCCGATTTCAGAAAGGATTTTCAGTTCTACAAGGTGAGGGAAATTAATA
ACTACCATCACGCTCACGATGCATATTTGAATGCTGTTGTGGGAACAGCACTCATTAAGAAATACCCAAAACTTGA
ATCTGAGTTCGTTTACGGAGATTACAAGGTTTACGATGTGAGAAAAATGATCGCTAAGTCTGAACAAGAGATTGG
TAAAGCTACTGCAAAGTACTTTTTCTATTCAAACATTATGAACTTTTTCAAGACTGAAATCACATTGGCTAACGGA
GAGATAAGAAAGAGGCCTCTTATCGAAACTAATGGTGAAACAGGAGAGATTGTTTGGGATAAAGGTAGAGATTTC
GCAACTGTTAGGAAGGTGCTTTCTATGCCACAGGTTAACATAGTGAAGAAAACTGAAGTTCAAACAGGAGGTTTT
TCTAAGGAGTCAATCTTGCCTAAGAGAAACTCTGATAAGTTAATCGCTAGGAAGAAAGATTGGGATCCAAAGAAA
TACGGAGGTTTTGATAGTCCTACAGTTGCTTATTCCGTGTTGGTTGTGGCAAAGGTTGAAAAGGGAAAATCTAAGA
AACTTAAGTCAGTGAAGGAGCTTTTGGGTATCACTATCATGGAAAGATCTTCATTTGAGAAAAACCCAATCGATTT
CTTGGAAGCTAAAGGATACAAGGAGGTTAAGAAAGATTTGATCATTAAATTACCTAAGTATTCATTATTCGAACTC
GAGAATGGTAGAAAAAGGATGCTCGCTAGTGCAGGAGAACTTCAAAAGGGTAACGAGTTAGCTCTCCCATCCAAG
TATGTTAATTTTCTTTACTTGGCAAGTCATTACGAAAAACTTAAAGGTTCCCCTGAAGATAATGAGCAAAAACAGT
TGTTCGTTGAACAGCATAAGCACTACCTTGATGAAATTATTGAGCAAATCAGTGAGTTTTCCAAAAGAGTTATTTT
GGCTGATGCAAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCATAGAGATAAGCCAATAAGGGAACAAGCAGA
GAACATAATCCACCTTTTTACTTTGACAAATCTTGGTGCTCCAGCTGCTTTTAAGTACTTCGATACTACAATCGATA
GAAAAAGGTACACTTCAACAAAGGAAGTTTTGGATGCAACTCTCATACATCAGAGTATCACTGGTCTTTATGAGAC
AAGAATTGATCTCTCCCAACTTGGAGGAGATAAAAGGCCAGCAGCAACTAAGAAAGCAGGTCAAGCAAAGAAAA
AGAAGCCAAAGAAGAAGAGAAAGGTTTGAtcccccgggggcgtcgacgtcttggccgaatttccccgatcgttcaaacatttggcaataaagtttcttaagattg
aatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatac
atttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcg
Online Resource 8 The complete DNA sequence of tobacco codon-optimized Cas9 expression vector.
The 2×35S promoter is shown in red lower case characters, the tobacco codon-optimized Cas9 in black
upper case characters, and the Nos terminator in green lower case characters.