刘耀光 - 多靶点pCRISPR载体（单子叶和双子叶植物用）使用方法2015-4-14(new)(1)

附录3：载体序列

GenBank data library under accession numbers: KR029097, KR029098, KR029099, KR0290100, KR0290101, KR0290102, KR0290103, KR0290104, KR0290105, KR0290106, KR0290107, KR029108 for the CRISPR/sgRNA intermediate plasmids, and KR0290109, KR0290110, KR0290111, KR0290112, KR0290113 for the CRISPR/Cas9 binary vectors.

LacZ-OsU6a-sgRNA structure in the plasmidBsaIBamHIacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGACGCGTTGAMluIE.coliPromoter LacZ(alpha)CATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaa tcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagtt gcgcagcctgaatggctaaTTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCGAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone）BsaIHindIII

PCR product of LacZ-U6a-sgRNA (831 bp, 801 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaaTTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

OsU6a-sgRNA structure in the plasmidBsaIBamHIacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCGAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone）Hind IIIBsaI

PCR product of OsU6a-sgRNA (629 bp, 599 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAG

AGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCG(19-20 bp target)GTTTTAG

AGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT GGAGTGGATGGNNNNNNNCGAGACCCACGCT

OsU6b-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGATGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGAGAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGCCCACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGGAGACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTGAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

PCR product of OsU6b-sgRNA (515 bp, 485 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGATGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGAGAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGCCGACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGGAGACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACG CT

OsU6c-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGActcattagcggtatgcatgttggtagaagtcggagatgtaaataattttcattatataaaaaaggtacttcgagaaaaataaatgcatacgaattaattctttttatgttttttaaaccaagtatatagaatttattgatggttaaaatttcaaaaatatgacgagagaaaggttaaacgtacggcatatacttctgaacagagagggaatatggggtttttgttgctcccaacaattcttaagcacgtaaaggaaaaaagcacattatccacattgtacttccagagatatgtacagcattacgtaggtacgttttctttttcttcccggagagatgatacaataatcatgtaaacccagaatttaaaaaatattctttactataaaaattttaattagggaacgtattattttttacatgacaccttttgagaaagagggacttgtaatatgggacaaatgaacaatttctaagaaatgggcatatgactctcagtacaatggaccaaattccctccagtcggcccagcaatacaaagggaaagaaatgagggggcccacaggccacggcccacttttctccgtggtggggagatccagctagaggtccggcccacaagtggcccttgccccgtgggacggtgggattgcagagcgcgtgggcggaaacaacagtttagtaccacctcgctcacgcaacgacgcgaccacttgcttataagctgctgcgctgaggctcagAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

PCR product of OsU6c-gRNA (924 bp, 894 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGActcattagcggtatgcatgttggtagaagtcggagatgtaaataattttcattatataaaaaaggtacttcgagaaaaataaatgcatacgaattaattctttttatgttttttaaaccaagtatatagaatttattgatggttaaaatttcaaaaatatgacgagagaaaggttaaacgtacggcatatacttctgaacagagagggaatatggggtttttgttgctcccaacaattcttaagcacgtaaaggaaaaaagcacattatccacattgtacttccagagatatgtacagcattacgtaggtacgttttctttttcttcccggagagatgatacaataatcatgtaaacccagaatttaaaaaatattctttactataaaaattttaattagggaacgtattattttttacatgacaccttttgagaaagagggacttgtaatatgggacaaatgaacaatttctaagaaatgggcatatgactctcagtacaatggaccaaattccctccagtcggcccagcaatacaaagggaaagaaatgagggggcccacaggccacggcccacttttctccgtggtggggagatccagctagaggtccggcccacaagtggcccttgccccgtgggacggtgggattgcagagcgcgtgggcggaaacaacagtttagtaccacctcgctcacgcaacgacgcgaccacttgcttataagctgctgcgctgaggctcaG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

TGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

LacZ-OsU3-sgRNA structure in the plasmid

PCR product of LacZ-OsU3-sgRNA (766 bp, 736 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTATGctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcTAAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA (19-20 target）GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

OsU3-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCAAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

PCR product of OsU3-sgRNA (603 bp, 573 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA(19-20 bp target) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG

GCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCC ACGCT

LacZ-AtU3b-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaatttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

PCR product of LacZ-AtU3b-sgRNA (709 bp, 679 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaatttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcA(19-20 bp target)GTTTTAGAGCTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGT GGATGGNNNNNNNCGAGACCCACGCT

AtU3b-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAtttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

PCR product of LacZ-AtU3b-sgRNA (507 bp, 477 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAtttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcA(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

PCR product of LacZ-AtU3d-sgRNA (486 bp, 456 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgc

tggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaaataagcttatgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtca (19-20 bp target) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG TGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

AtU3d-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAataagctt atgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone）

PCR product of AtU3d-sgRNA (284 bp, 254 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAataagcttatgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtcA(19-20 bp target)GTTTTAGAGCTA GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGT GGATGGNNNNNNNCGAGACCCACGCT

PCR product of AtU6-1-sgRNA (487 bp, 457 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAAGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAGAGACGAAGTAGTGATTG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

AtU6-29-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAaaatatcagagatctcttacagttagtttcgttcttaatccaaactactgcagcctgacagacaaatgaggatgcaaacaattttaaagtttatctaacgctagctgttttgtttcttctctctggtgcaccaacgacggcgttttctcaatcataaagaggcttgttttacttaaggccaataatgttgatggatcgaaagaagagggcttttaataaacgagcccgtttaagctgtaaacgatgtcaaaaacatcccacatcgttcagttgaaaatagTagctctgtttatatattggtagagtcgactaagagattgAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)PCR product of AtU6-29-sgRNA (502 bp, 472 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAaaatatcagagatctcttacagttagtttcgttcttaatccaaactactgcagcctgacagacaaatgaggatgcaaacaattttaaagtttatctaacgctagctgttttgtttcttctctctggtgcaccaacgacggcgttttctcaatcataaagaggcttgttttacttaaggccaataatgttgatggatcgaaagaagagggcttttaataaacgagcccgtttaagctgtaaacgatgtcaaaaacatcccacatcgttcagttgaaaatagTagctctgtttatatattggtagagtcgactaagagattG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT