CRISPRCas9-mediated targeted mutagenesi
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: 1State Key Laboratory of Silkworm Genome Biology, Southwest University,
Chongqing 400715, China
2School of Life Science, Chongqing University, Chongqing 400030, China
?These authors contributed equally to this work.
*To whom correspondence should be addressed:
Email: xiaqy@https://www.360docs.net/doc/3e2612973.html,
Online Resource 1 List of primers used in this study.
Online Resource 2Targeted mutagenesis on the NtPDR6gRNA2 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 3Targeted 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 4Targeted 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 5Off-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).
AGCTTTCGTTTTCTTCTTTTTAACTTTCCA TTCGGAGTTTTTGTATCTTGTTTCATAGTTTGTCCCAGGA TTAGAATGA T TAGGCATCGAACCTTCAAGAATTTGATTGAA TAAAACATCTTCATTCTTAAGATA TGAAGATAATCTTCAAAAGGCCC CTGGGAATCTGAAAGAAGAGAAGCAGGCCCA TTTA TATGGGAAAGAACAA TAGTA TTTCTTA TATAGGCCCA TTTAAG TTGAAAACAA TCTTCAAAAGTCCCACA TCGCTTAGATAAGAAAACGAAGCTGAGTTTATATACAGCTAGAGTCGAAG TAGTGATT NNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGCAAGTTAAAA TAAGGCTAGTCCGTTA TCA ACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT GCAAAATTTTCCAGATCGATTTCTTCTTCCTCTGTTCTTCGGC 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 aaattttcaccatttacgaacgata ctcgagggggggtaccccccaatacttgttgtggactagtcgagctcgATGGATTACAAGGATCACGATGGAGATTACA 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 AGAAGCCAAAGAAGAAGAGAAAGGTTTGAtcccccgggggcgtcgacgtcttggcc gaatttccccgatcgttcaaacatttggcaataaagtttcttaagattg 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.