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Articleshttps://doi.org/10.1038/s41587-020-0566-4
Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC–Cas9Shengxing Wang1,4, Yuan Zong1,4, Qiupeng Lin1,2,4, Huawei Zhang1,4, Zhuangzhuang Chai1,2, Dandan Zhang2,3, Kunling Chen1, Jin-Long Qiu 2,3 and Caixia Gao 1,2 ✉
1State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China. 2College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China. 3State Key Laboratory of Plant Genomics, Institute of Microbiology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China. 4These authors contributed equally: Shengxing Wang, Yuan Zong, Qiupeng Lin, Huawei Zhang. ✉e-mail: [email protected]
SUPPLEMENTARY INFORMATION
In the format provided by the authors and unedited.
NAturE BiOtECHNOLOGY | www.nature.com/naturebiotechnology
Supplementary Figure 1 Comparison of insertion/deletion mutation types induced by
Cas9 and AFIDs 1-3 at two target sites in rice protoplasts. (a, b) The mutation types
generated by Cas9 and AFIDs 1-3 at the target sites of OsAAT (a) and OsCDC48-T1
(b). Each displayed mutation type was filtered by > 2% of indels with two or more
repeat experiments. Proportions of indels (mean) were calculated from three
biologically independent experiments (n = 3), and proportions of predictable deletions
were marked in red.
Supplementary Figure 2 Comparison of insertion/deletion mutation types generated
by Cas9 and AFIDs 1-3 at four target sites in wheat protoplasts. (a-d) The mutation
types generated by Cas9 and AFIDs 1-3 at the target sites of TaF3H (a), TaGASR6 (b),
TaPMK (c), and TaVRN1 (d). Each mutation type displayed was filtered by > 2% of
indels with two or more repeat experiments. Proportions of indels (mean) were
calculated from three biologically independent experiments (n = 3), and proportions of
predictable deletions were marked in red.
Supplementary Figure 3 Comparison of C-to-T conversion rates by A3A-PBE in
different deamination positions at eight tested target sites in rice and wheat protoplasts.
(a-h) The C-to-T conversion rates generated by A3A-PBE at the target sites of OsAAT
(a), OsCDC48-T1 (b), OsNRT1.1B (c), TaF3H (d), TaGASR6 (e), TaMYB10 (f), TaPMK
(g), and TaVRN1 (h). Frequencies (mean ± s.e.m.) were calculated from three
biologically independent experiments (n = 3).
Supplementary Figure 4 AFID-3 induces predictable deletions in miRNA genes and
cis-acting elements in wheat protoplasts. (a) Comparison of deletion frequencies
induced by Cas9 and AFID-3 at target sites of TamiR160, TamiR319, TamiR396,
TamiR444a genes and putative G-box of TaVRN1-B1, putative TALE-BE and NAC-BE
of TaPDS-A1. Significant differences between Cas9 and AFID-3 were tested by two-
tailed Student’s t-tests. (b-h) Comparison of the insertion/deletion mutation types
induced by Cas9 and AFID-3 at target sites in TamiR160 (b), TamiR319 (c), TamiR396
(d), TamiR444a (e), NAC-BE of TaPDS-A1 (f), G-box of TaVRN1-B1 (g) and TALE-
BE of TaPDS-A1 (h). Each mutation type shown was filtered by > 2% of indels with
two or more repeat experiments. Frequencies (mean ± s.e.m.) and proportions (mean)
were calculated from three biologically independent experiments (n = 3) in a-h, and
proportions of predictable deletions were marked in red.
Supplementary Figure 5 Characterization of 1-nucleotide template-dependent
insertions among AFID-3-generated deletions. (a) Proportions of AFID-3-generated
predictable deletion types, and different deletion types harboring 1-bp templated
insertions, at the 5’-deamination site, 3’-Cas9-cleavage site, and both sites, compared
with predictable deletion types at 15 tested target sites. Proportions (mean) were
calculated from three biologically independent experiments (n = 3). (b) Possible
alternative end processing of AFID-3-generated deletions resulting in the 1-bp
templated insertions at the 5’-deamination and 3’-Cas9-cleavage sites. The green oval
represents DNA polymerase, and the red and purple arrowheads indicate the possible
cleavage positions of the RuvC and HNH domains, and the Artemis-like nuclease,
respectively.
Supplementary Figure 6 The detail mutation types generated by AFID-3 at the target
site of TamiR396 in wheat regenerated plants. The red box represented the predictable
deletion type.
Supplementary Figure 7 Comparison of mutation types generated by Cas9 and AFID-
3 at the target site of OsCDC48-T2 in rice regenerated plants. (a) Architectures of the
binary vectors pH-Cas9 and pH-AFID-3. (b) Part of indel mutants induced by Cas9 and
AFID-3 at the target site of OsCDC48-T2. The red box represented the predictable
deletion type.
Supplementary Figure 8 Comparison of deletion types from the deaminated Cs to the
DSB induced by AFID-3 and eAFID-3 in rice protoplasts. (a-e) The mutation types
generated by AFID-3 and eAFID-3 at the target sites of OsAAT (a), OsCDC48-T2 (b),
OsNRT1.1B (c), OsPDS (d), OsSPL14 (e). The green and light red bars represent the
deletion type extending from TC motif and other motifs (AC, CC, and GC) to the DSB,
respectively. Each mutation type shown was filtered by > 2% of indels with two or more
repeat experiments. Proportions (mean ± s.e.m.) were calculated from three biologically
independent experiments (n = 3).
Supplementary Figure 9 Comparison of predictable in-frame deletion types induced
by Cas9, AFID-3, and eAFID-3 at the miRNA-binding sites in rice protoplasts. (a, b)
The mutation types generated by Cas9, AFID-3, and eAFID-3 at miR156-binding site
of OsIPA1 (a) and miR396h-binding site of OsGRF1 (b) in rice protoplasts. The green
and light red bars represent the deletion type extending from TC motif and other motifs
(AC, CC, and GC) to the DSB, respectively. Each mutation type shown was filtered by >
2% of indels with two or more repeat experiments. Proportions (mean ± s.e.m.) were
calculated from three biologically independent experiments (n = 3). The red box
represented the predictable in-frame deletions.
Supplementary Table 1. Description of sgRNA target sites and sequences sgRNA Target sequence Oligo-F Oligo-R
sgOsAAT CAAGGATCCCAGCCCCGTGAAGG GGCGCAAGGATCCCAGCCCCGTGA AAACTCACGGGGCTGGGATCCTTG sgOsACC TCCACAGCTATCACACCCACTGG GGCGTCCACAGCTATCACACCCAC AAACGTGGGTGTGATAGCTGTGGA sgOsCDC48-T1 GACCAGCCAGCGTCTGGCGCCGG GGCGACCAGCCAGCGTCTGGCGC AAACGCGCCAGACGCTGGCTGGT sgOsCDC48-T2 AAGGCAGGGTCAATGATATCTGG GGCGAAGGCAGGGTCAATGATATC AAACGATATCATTGACCCTGCCTT sgOsEV CACACACACACTAGTACCTCTGG GGCGCACACACACACTAGTACCTC AAACGAGGTACTAGTGTGTGTGTG sgOsGRF1-miRT GAACCGTTCAAGAAAGCCTGTGG GGCGAACCGTTCAAGAAAGCCTG AAACCAGGCTTTCTTGAACGGTT sgOsIPA1-miRT CTCTTCTGTCAACCCAGCCATGG GGCGCTCTTCTGTCAACCCAGCCA AAACCTCTTCTGTCAACCCAGCCA sgOsNRT1.1B ACTAGATATCTAAACCATTAAGG GGCGACTAGATATCTAAACCATTA AAACTAATGGTTTAGATATCTAGT sgOsPDS GTTGGTCTTTGCTCCTGCAGAGG GGCGTTGGTCTTTGCTCCTGCAG AAACCTGCAGGAGCAAAGACCAA sgOsSPL14 CCACCGGAGATCCGATCGCCTGG GGCGCCACCGGAGATCCGATCGCC AAACGGCGATCGGATCTCCGGTGG sgOsSWEET14 GCTTAGCACCTGGTTGGAGGGGG GGCGCTTAGCACCTGGTTGGAGG AAACCCTCCAACCAGGTGCTAAG sgTaF3H CGCCACGCGGTCCCGGATCTCGG CTTGCGCCACGCGGTCCCGGATCT AAACAGATCCGGGACCGCGTGGCG sgTaGASR6 TCCTCGTTGCCGGCGGTGCCGGG CTTGTCCTCGTTGCCGGCGGTGCC AAACGGCACCGCCGGCAACGAGGA sgTaMYB10 TGGCTCAACTACCTCCGGCCGGG CTTGTGGCTCAACTACCTCCGGCC AAACGGCCGGAGGTAGTTGAGCCA sgTaPMK CGCGGCGTCGAACCCGGCCGTGG CTTGCGCGGCGTCGAACCCGGCCG AAACCGGCCGGGTTCGACGCCGCG sgTaVRN1 CCTTCTCCAAGCGCCGCTCGGGG CTTGCCTTCTCCAAGCGCCGCTCG AAACCGAGCGGCGCTTGGAGAAGG sgTamiR160 CGAGTGGCATACAGGGAGCCAGG CTTGCGAGTGGCATACAGGGAGCC AAACGGCTCCCTGTATGCCACTCG sgTamiR319 CTCCTCCATGCTTGGACTGAAGG CTTGCTCCTCCATGCTTGGACTGA AAACTCAGTCCAAGCATGGAGGAG sgTamiR396 ACTGTGAACTCGCGGGGATGGGG CTTGACTGTGAACTCGCGGGGATG AAACCATCCCCGCGAGTTCACAGT sgTamiR444a AAAGGGAGGCAGCAAGCTTGAGG CTTGAAAGGGAGGCAGCAAGCTTG AAACCAAGCTTGCTGCCTCCCTTT sgG-box TGGGCTATAACACGTAAAACAGG CTTGTGGGCTATAACACGTAAAAC AAACGTTTTACGTGTTATAGCCCA sgTALE-BE GACGCAGCGGCGAGGCGAGGCGG CTTGACGCAGCGGCGAGGCGAGG AAACCCTCGCCTCGCCGCTGCGT sgNAC-BE GGCTGCACGGTAAGCAAATCGGG CTTGGCTGCACGGTAAGCAAATC AAACGATTTGCTTACCGTGCAGC
The PAM motif in each target sequence is shown in bold. Oligo-F and Oligo-R were used for preparing the sgRNA.
Supplementary Table 2. PCR primers used in this study Primer name Forward primer Reverse primer Application
OsAAT TCGTCCGTCTTCGCTGGCC GAACACGGAATCTGTACACTCAGCC 1st PCR for deep sequencing of OsAAT target site
OsAAT-seq1 CGATGTTCGACCTGATCGGTGCTC TGACCAATCCACCACCAATCCAATCC
2nd PCR for deep sequencing of OsAAT target site
OsAAT-seq2 ACAGTGTCGACCTGATCGGTGCTC GCCAATATCCACCACCAATCCAATCC OsAAT-seq3 CAGATCTCGACCTGATCGGTGCTC CTTGTAATCCACCACCAATCCAATCC OsAAT-seq4 ATCACGTCGACCTGATCGGTGCTC TTAGGCATCCACCACCAATCCAATCC OsAAT-seq5 ACTTGATCGACCTGATCGGTGCTC GATCAGATCCACCACCAATCCAATCC OsAAT-seq6 TAGCTTTCGACCTGATCGGTGCTC GGCTACATCCACCACCAATCCAATCC OsAAT-seq7 AGTCAATCGACCTGATCGGTGCTC AGTTCCATCCACCACCAATCCAATCC OsAAT-seq8 ATGTCATCGACCTGATCGGTGCTC CCGTCCATCCACCACCAATCCAATCC OsAAT-seq9 GTAGAGTCGACCTGATCGGTGCTC GTCCGCATCCACCACCAATCCAATCC OsAAT-seq10 GTGAAATCGACCTGATCGGTGCTC GTGGCCATCCACCACCAATCCAATCC
OsACC TTCTTGGGCGGGAAGTGTA AGGCAGGCTGATTGTATGTC 1st PCR for deep sequencing of OsACC target site
OsACC-seq1 CGATGTTAAAGACAGCTTTGTGGAAAC TGACCACAGCACGAGGAACAGATTG
2nd PCR for deep sequencing of OsACC target site
OsACC-seq2 ACAGTGTAAAGACAGCTTTGTGGAAAC GCCAATCAGCACGAGGAACAGATTG OsACC-seq3 CAGATCTAAAGACAGCTTTGTGGAAAC CTTGTACAGCACGAGGAACAGATTG OsACC-seq4 ATCACGTAAAGACAGCTTTGTGGAAAC TTAGGCCAGCACGAGGAACAGATTG OsACC-seq5 ACTTGATAAAGACAGCTTTGTGGAAAC GATCAGCAGCACGAGGAACAGATTG OsACC-seq6 TAGCTTTAAAGACAGCTTTGTGGAAAC GGCTACCAGCACGAGGAACAGATTG OsACC-seq7 AGTCAATAAAGACAGCTTTGTGGAAAC AGTTCCCAGCACGAGGAACAGATTG OsACC-seq8 ATGTCATAAAGACAGCTTTGTGGAAAC CCGTCCCAGCACGAGGAACAGATTG OsACC-seq9 GTAGAGTAAAGACAGCTTTGTGGAAAC GTCCGCCAGCACGAGGAACAGATTG
OsACC-seq10 GTGAAATAAAGACAGCTTTGTGGAAAC GTGGCCCAGCACGAGGAACAGATTG
OsCDC48T1 CCTCTGCCTGATGATCAATCTAGGC TCATCATCGTCAGCTGCGGC 1st PCR for deep sequencing of OsCDC48-T1 target site
OsCDC48T1-seq1 CGATGTCGACATCCGCAAGTACCAG TGACCAAAATCATCATCGTCAGCTGC
2nd PCR for deep sequencing of OsCDC48-T1 target site
OsCDC48T1-seq2 ACAGTGCGACATCCGCAAGTACCAG GCCAATAAATCATCATCGTCAGCTGC OsCDC48T1-seq3 CAGATCCGACATCCGCAAGTACCAG CTTGTAAAATCATCATCGTCAGCTGC OsCDC48T1-seq4 AGTCAACGACATCCGCAAGTACCAG AGTTCCAAATCATCATCGTCAGCTGC OsCDC48T1-seq5 GTAGAGCGACATCCGCAAGTACCAG GTCCGCAAATCATCATCGTCAGCTGC OsCDC48T1-seq6 GTTTCGCGACATCCGCAAGTACCAG CGTACGAAATCATCATCGTCAGCTGC OsCDC48T1-seq7 ACTGATCGACATCCGCAAGTACCAG ATGAGCAAATCATCATCGTCAGCTGC OsCDC48T1-seq8 CAACTACGACATCCGCAAGTACCAG CACCGGAAATCATCATCGTCAGCTGC OsCDC48T1-seq9 CAGGCGCGACATCCGCAAGTACCAG CATGGCAAATCATCATCGTCAGCTGC OsCDC48T1-seq10 CGGAATCGACATCCGCAAGTACCAG CTAGCTAAATCATCATCGTCAGCTGC
OsCDC48T2 GGCAGTCAGCTCCTTGTG GTCCTTCCTCCGCTTCTC 1st PCR for deep sequencing of OsCDC48-T2 target site
OsCDC48T2-seq1 CGATGTTGCTGATAGAGTGCTGAAC TGACCAGGAGGCAAGCTTTGAAGA
2nd PCR for deep sequencing of OsCDC48-T2 target site
OsCDC48T2-seq2 ACAGTGTGCTGATAGAGTGCTGAAC GCCAATGGAGGCAAGCTTTGAAGA OsCDC48T2-seq3 CAGATCTGCTGATAGAGTGCTGAAC CTTGTAGGAGGCAAGCTTTGAAGA OsCDC48T2-seq4 ATCACGTGCTGATAGAGTGCTGAAC TTAGGCGGAGGCAAGCTTTGAAGA OsCDC48T2-seq5 ACTTGATGCTGATAGAGTGCTGAAC GATCAGGGAGGCAAGCTTTGAAGA OsCDC48T2-seq6 TAGCTTTGCTGATAGAGTGCTGAAC GGCTACGGAGGCAAGCTTTGAAGA OsCDC48T2-seq7 AGTCAATGCTGATAGAGTGCTGAAC AGTTCCGGAGGCAAGCTTTGAAGA OsCDC48T2-seq8 ATGTCATGCTGATAGAGTGCTGAAC CCGTCCGGAGGCAAGCTTTGAAGA OsCDC48T2-seq9 GTAGAGTGCTGATAGAGTGCTGAAC GTCCGCGGAGGCAAGCTTTGAAGA OsCDC48T2-seq10 GTGAAATGCTGATAGAGTGCTGAAC GTGGCCGGAGGCAAGCTTTGAAGA
OsEV GGGAGATGAGAGAGCTTGTGCC GAGTAGTGTAGTACTGAAGAAGCACAGC 1st PCR for deep sequencing of OsEV target site
OsEV-seq1 CGATGTCCATCCATTCATTCCATCTCT TGACCAGAGACCATGAAAGAGAGAGAGAG
2nd PCR for deep sequencing of OsEV target site
OsEV-seq2 ACAGTGCCATCCATTCATTCCATCTCT GCCAATGAGACCATGAAAGAGAGAGAGAG OsEV-seq3 CAGATCCCATCCATTCATTCCATCTCT CTTGTAGAGACCATGAAAGAGAGAGAGAG OsEV-seq4 AGTCAACCATCCATTCATTCCATCTCT AGTTCCGAGACCATGAAAGAGAGAGAGAG OsEV-seq5 GTAGAGCCATCCATTCATTCCATCTCT GTCCGCGAGACCATGAAAGAGAGAGAGAG OsEV-seq6 GTTTCGCCATCCATTCATTCCATCTCT CGTACGGAGACCATGAAAGAGAGAGAGAG OsEV-seq7 ACTGATCCATCCATTCATTCCATCTCT ATGAGCGAGACCATGAAAGAGAGAGAGAG OsEV-seq8 CAACTACCATCCATTCATTCCATCTCT CACCGGGAGACCATGAAAGAGAGAGAGAG OsEV-seq9 CAGGCGCCATCCATTCATTCCATCTCT CATGGCGAGACCATGAAAGAGAGAGAGAG OsEV-seq10 CGGAATCCATCCATTCATTCCATCTCT CTAGCTGAGACCATGAAAGAGAGAGAGAG
OsNRT1.1B AGCTAGGAGTAGAGAACGAGACATATAC GTTGGGAGAATAGCTGAAGCTATCGG 1st PCR for deep sequencing of OsNRT1.1B target site
OsNRT1.1B-seq1 CGATGTACTCCAGCCACTCACTGTC TGACCAGTCGTTGGGACTGGGCTAC
2nd PCR for deep sequencing of OsNRT1.1B target site
OsNRT1.1B-seq2 ACAGTGACTCCAGCCACTCACTGTC GCCAATGTCGTTGGGACTGGGCTAC OsNRT1.1B-seq3 CAGATCACTCCAGCCACTCACTGTC CTTGTAGTCGTTGGGACTGGGCTAC OsNRT1.1B-seq4 ATCACGACTCCAGCCACTCACTGTC TTAGGCGTCGTTGGGACTGGGCTAC OsNRT1.1B-seq5 ACTTGAACTCCAGCCACTCACTGTC GATCAGGTCGTTGGGACTGGGCTAC OsNRT1.1B-seq6 GTTTCGACTCCAGCCACTCACTGTC CGTACGGTCGTTGGGACTGGGCTAC OsNRT1.1B-seq7 GAGTGGACTCCAGCCACTCACTGTC GGTAGCGTCGTTGGGACTGGGCTAC OsNRT1.1B-seq8 ACTGATACTCCAGCCACTCACTGTC ATGAGCGTCGTTGGGACTGGGCTAC OsNRT1.1B-seq9 ATTCCTACTCCAGCCACTCACTGTC CAAAAGGTCGTTGGGACTGGGCTAC OsNRT1.1B-seq10 CAACTAACTCCAGCCACTCACTGTC CACCGGGTCGTTGGGACTGGGCTAC
OsSWEET14 GAGGACAGAGTTATGAAGGAAACA GGAGACCAAAGGCGAAGG Amplifying the OsSWEET14 target site for mutant detection
OsPDS CAAAAGGCCCAAAAGTGC GGCAAGGTTCACAGTCCG 1st PCR for deep sequencing of OsPDS target site
OsPDS-seq1 CGATGTACGGTTTTCTTTCCTTTTGT TGACCAATCAGCAGCAATTTCATCAG
2nd PCR for deep sequencing of OsPDS target site
OsPDS-seq2 ACAGTGACGGTTTTCTTTCCTTTTGT GCCAATATCAGCAGCAATTTCATCAG OsPDS-seq3 CAGATCACGGTTTTCTTTCCTTTTGT CTTGTAATCAGCAGCAATTTCATCAG OsPDS-seq4 ATCACGACGGTTTTCTTTCCTTTTGT TTAGGCATCAGCAGCAATTTCATCAG OsPDS-seq5 ACTTGAACGGTTTTCTTTCCTTTTGT GATCAGATCAGCAGCAATTTCATCAG
OsSPL14 ATCACGCTACGGTCGACTAGCTG TGAGGTTCTTCAGTTCCATGACTC 1st PCR for deep sequencing of OsSPL14 target site
OsSPL14-seq1 CGATGTAGAGCATCGCAGGTTCAGA TGACCAGCTACCTTGGCCGCTGTAT
2nd PCR for deep sequencing of OsSPL14 target site
OsSPL14-seq2 ACAGTGAGAGCATCGCAGGTTCAGA GCCAATGCTACCTTGGCCGCTGTAT OsSPL14-seq3 CAGATCAGAGCATCGCAGGTTCAGA CTTGTAGCTACCTTGGCCGCTGTAT OsSPL14-seq4 ATCACGAGAGCATCGCAGGTTCAGA TTAGGCGCTACCTTGGCCGCTGTAT OsSPL14-seq5 ACTTGAAGAGCATCGCAGGTTCAGA GATCAGGCTACCTTGGCCGCTGTAT
OsGRF1-miRT GTTGCCGATGCTAACAAAGTCCA CCATGCCGAGCTGCTTGAACT 1st PCR for deep sequencing of miR396h-binding site in OsGRF1
OsGRF1-miRT-seq1 CGATGTGTACGGACGGCAAGAAGTGG TGACCACCGGCGGAGGTGTTCGACG
2nd PCR for deep sequencing of miR396h-binding site in OsGRF1
OsGRF1-miRT-seq2 ACAGTGGTACGGACGGCAAGAAGTGG GCCAATCCGGCGGAGGTGTTCGACG OsGRF1-miRT-seq3 CAGATCGTACGGACGGCAAGAAGTGG CTTGTACCGGCGGAGGTGTTCGACG OsGRF1-miRT-seq4 ATCACGGTACGGACGGCAAGAAGTGG TTAGGCCCGGCGGAGGTGTTCGACG OsGRF1-miRT-seq5 ACTTGAGTACGGACGGCAAGAAGTGG GATCAGCCGGCGGAGGTGTTCGACG
OsIPA1-miRT GGTTGCATTCGCTGATGTGT GAGAAGTGGGCATGATGGCT 1st PCR for deep sequencing of miR156-binding site in OsIPA1
OsIPA1-miRT-seq1 CGATGTGTAGCTCTTCTTCAGGGCCACC TGACCAACAGGATTGCCATCAAAGCTGG
2nd PCR for deep sequencing of miR156-binding site in OsIPA1
OsIPA1-miRT-seq2 ACAGTGGTAGCTCTTCTTCAGGGCCACC GCCAATACAGGATTGCCATCAAAGCTGG OsIPA1-miRT-seq3 CAGATCGTAGCTCTTCTTCAGGGCCACC CTTGTAACAGGATTGCCATCAAAGCTGG OsIPA1-miRT-seq4 ATCACGGTAGCTCTTCTTCAGGGCCACC TTAGGCACAGGATTGCCATCAAAGCTGG OsIPA1-miRT-seq5 ACTTGAGTAGCTCTTCTTCAGGGCCACC GATCAGACAGGATTGCCATCAAAGCTGG
TaF3H TCGATACGGCTGGATGGATG GGTGGCTGGAGACGATGAAG 1st PCR for deep sequencing of TaF3H target site
TaF3H-seq1 CGATGTTCCTTCGTGCGGGACGAGGA TGACCAATGTCGGCGATGAGGTCGG
2nd PCR for deep sequencing of TaF3H target site
TaF3H-seq2 CAGATCTCCTTCGTGCGGGACGAGGA CTTGTAATGTCGGCGATGAGGTCGG TaF3H-seq3 ACTTGATCCTTCGTGCGGGACGAGGA GATCAGATGTCGGCGATGAGGTCGG TaF3H-seq4 TAGCTTTCCTTCGTGCGGGACGAGGA GGCTACATGTCGGCGATGAGGTCGG TaF3H-seq5 CACGATTCCTTCGTGCGGGACGAGGA CACTCAATGTCGGCGATGAGGTCGG
TaGASR6 CTTGCTTGCATTATTCT TGCTCATGTTATGTGGA 1st PCR for deep sequencing of TaGASR6 target site
TaGASR6-seq1 CGATGTTGCTCGTACCGGTGCAGCAA TGACCATCATGGGCACTTGTGGCGG
2nd PCR for deep sequencing of TaGASR6 target site
TaGASR6-seq2 CAGATCTGCTCGTACCGGTGCAGCAA CTTGTATCATGGGCACTTGTGGCGG TaGASR6-seq3 ACTTGATGCTCGTACCGGTGCAGCAA GATCAGTCATGGGCACTTGTGGCGG TaGASR6-seq4 TAGCTTTGCTCGTACCGGTGCAGCAA GGCTACTCATGGGCACTTGTGGCGG TaGASR6-seq5 CACGATTGCTCGTACCGGTGCAGCAA CACTCATCATGGGCACTTGTGGCGG
TaMYB10 GTCTCCTACATCAACGAT GCTGACTGATATGGATAGTA 1st PCR for deep sequencing of TaMYB10 target site
TaMYB10-seq1 CGATGTGCATAGCTCGAGATGGTCGTG TGACCAGTGCACCTGTTGCCTAGGAGC 2nd PCR for deep sequencing of TaMYB10 target site
TaMYB10-seq2 CAGATCGCATAGCTCGAGATGGTCGTG CTTGTAGTGCACCTGTTGCCTAGGAGC TaMYB10-seq3 ACTTGAGCATAGCTCGAGATGGTCGTG GATCAGGTGCACCTGTTGCCTAGGAGC TaMYB10-seq4 TAGCTTGCATAGCTCGAGATGGTCGTG GGCTACGTGCACCTGTTGCCTAGGAGC
TaMYB10-seq5 CACGATGCATAGCTCGAGATGGTCGTG CACTCAGTGCACCTGTTGCCTAGGAGC
TaPMK GCCGTGCTCCTGCTCTACCTCTG CACGTCGCTCTTCTCCGTCAGC 1st PCR for deep sequencing of TaPMK target site
TaPMK-seq1 CGATGTGCCACGGCCTGCACCACA TGACCATTGGTGATGGTGGCGGAGGAC
2nd PCR for deep sequencing of TaPMK target site
TaPMK-seq2 ACAGTGGCCACGGCCTGCACCACA GCCAATTTGGTGATGGTGGCGGAGGAC TaPMK-seq3 CAGATCGCCACGGCCTGCACCACA CTTGTATTGGTGATGGTGGCGGAGGAC TaPMK-seq4 ATCACGGCCACGGCCTGCACCACA TTAGGCTTGGTGATGGTGGCGGAGGAC TaPMK-seq5 ACTTGAGCCACGGCCTGCACCACA GATCAGTTGGTGATGGTGGCGGAGGAC
TaVRN1 TCGGGGCCAGATCCCTTTA CAAAATACGCCAGATGAACAACC 1st PCR for deep sequencing of TaVRN1 target site
TaVRN1-seq1 CGATGTCGGTCTGCCGGTGTTGGAG TGACCAGGAGAAGATGATGAGGCCGACC
2nd PCR for deep sequencing of TaVRN1 target site
TaVRN1-seq2 CAGATCCGGTCTGCCGGTGTTGGAG CTTGTAGGAGAAGATGATGAGGCCGACC TaVRN1-seq3 ACTTGACGGTCTGCCGGTGTTGGAG GATCAGGGAGAAGATGATGAGGCCGACC TaVRN1-seq4 TAGCTTCGGTCTGCCGGTGTTGGAG GGCTACGGAGAAGATGATGAGGCCGACC TaVRN1-seq5 CACGATCGGTCTGCCGGTGTTGGAG CACTCAGGAGAAGATGATGAGGCCGACC
TamiR160 CCAACACCTACCATTACCAAG AAGGAGGCTTAGATTGATTAG 1st PCR for deep sequencing of TamiR160 target site
TamiR160-seq1 CGATGTTCACGCCGTGCAATGCAG TGACCAAGAGAGAGAGAGACGGTCATGC 2nd PCR for deep sequencing of TamiR160 target site
TamiR160-seq2 ACAGTGTCACGCCGTGCAATGCAG GCCAATAGAGAGAGAGAGACGGTCATGC TamiR160-seq3 CAGATCTCACGCCGTGCAATGCAG CTTGTAAGAGAGAGAGAGACGGTCATGC
TamiR319 CTGCCGACTCATTCACTC TGATTAGCAAAGCCTCTG 1st PCR for deep sequencing of TamiR319 target site
TamiR319-seq1 CGATGTTACTGTGCGAATGAGCGAATG TGACCAGGGAAGTAGTACCAGAATCTGG 2nd PCR for deep sequencing of TamiR319 target site
TamiR319-seq2 ACAGTGTACTGTGCGAATGAGCGAATG GCCAATGGGAAGTAGTACCAGAATCTGG TamiR319-seq3 CAGATCTACTGTGCGAATGAGCGAATG CTTGTAGGGAAGTAGTACCAGAATCTGG
TamiR396 TAGCCTCCTCTTTCGGTGG GCGGAACTCAGAGCAAACA 1st PCR for deep sequencing of TamiR396 target site
TamiR396-seq1 CGATGTGAATTATGGCGTGGAAAGATGC TGACCACACAATCGGAACCCACATCG 2nd PCR for deep sequencing of TamiR396 target site
TamiR396-seq2 ACAGTGGAATTATGGCGTGGAAAGATGC GCCAATCACAATCGGAACCCACATCG TamiR396-seq3 CAGATCGAATTATGGCGTGGAAAGATGC CTTGTACACAATCGGAACCCACATCG
TamiR444a CGAGCATGAGGCAACAAC ATTAGCACAGTAAGCCAATC 1st PCR for deep sequencing of TamiR444a target site
TamiR444a-seq1 CGATGTCAAGGCGCAAAATTAATAGA TGACCACTAGATCAATTTTGTAGAACATG 2nd PCR for deep sequencing of TamiR444a target site
TamiR444a-seq2 ACAGTGCAAGGCGCAAAATTAATAGA GCCAATCTAGATCAATTTTGTAGAACATG TamiR444a-seq3 CAGATCCAAGGCGCAAAATTAATAGA CTTGTACTAGATCAATTTTGTAGAACATG
TaVRN1pro AACTAAATGCTGTTTTAGGCCCAACA AAGAATTAGACCGTCCATATAACTACGT 1st PCR for deep sequencing of G-box in TaVRN1
G-box-seq1 CGATGTGGAGACAGCCCAAAAC TGACCACACCAGCACTTAGCA 2nd PCR for deep sequencing of G-box in TaVRN1
G-box-seq2 ACAGTGGGAGACAGCCCAAAAC GCCAATCACCAGCACTTAGCA G-box-seq3 CAGATCGGAGACAGCCCAAAAC CTTGTACACCAGCACTTAGCA
TaPDSpro TGGAACCAGAAAGAAGACG TGTGAGATTCTTGTCAACCTTA 1st PCR for deep sequencing of TALE-BE and NAC-BE in TaPDS
TALE-BE-seq1 CGATGTCCTTATCCCGCGCCGTACATA TGACCAGGAAAATCCTCAACGCCCGC 2nd PCR for deep sequencing of TALE-BE in TaPDS
TALE-BE-seq2 ACAGTGCCTTATCCCGCGCCGTACATA GCCAATGGAAAATCCTCAACGCCCGC TALE-BE-seq3 CAGATCCCTTATCCCGCGCCGTACATA CTTGTAGGAAAATCCTCAACGCCCGC NAC-BE-seq1 CGATGTGGCGTTGAGGATTTTCCAAGGG TGACCATCCTAAGACCCCGTGTGTTTCC
2nd PCR for deep sequencing of NAC-BE in TaPDS
NAC-BE-seq2 ACAGTGGGCGTTGAGGATTTTCCAAGGG GCCAATTCCTAAGACCCCGTGTGTTTCC NAC-BE-seq3 CAGATCGGCGTTGAGGATTTTCCAAGGG CTTGTATCCTAAGACCCCGTGTGTTTCC
Supplementary Sequences. Complete coding sequences of AFID-3 and eAFID-3
fusion cistrons optimized in this study. The NLSs are written in lower cases. The codon-
optimized A3A/A3Bctd, XTEN linker, UDG, AP lyase, and P2A linker are highlighted
in red/green, blue, purple, orange, and gray, respectively. The codon-optimized Cas9 is
shown in bold. Supplementary Sequence 1: AFID-3 (A3A-Cas9-UDG-P2A-AP) ATGGAGGCCAGCCCGGCTAGCGGCCCAAGGCATCTCATGGACCCGCACATCTTCACCAGCAACTTCAACAACGGCATCGGCAGGCACAAGACCTACTTGTGCTACGAGGTGGAGAGGCTCGACAACGGAACCTCCGTGAAGATGGACCAACACAGGGGGTTCCTCCACAACCAAGCCAAGAACCTCCTCTGCGGCTTCTACGGCAGGCACGCCGAGTTGAGGTTCCTCGACTTGGTGCCATCCCTCCAACTCGATCCAGCCCAAATCTACCGCGTGACCTGGTTCATCTCCTGGTCCCCATGCTTCTCCTGGGGTTGCGCCGGCGAGGTTCGGGCTTTCCTCCAAGAAAACACCCACGTCCGCCTCCGCATTTTCGCCGCCAGGATCTATGATTACGACCCTCTCTACAAGGAGGCCCTCCAGATGCTGCGGGACGCCGGTGCTCAGGTGAGTATCATGACCTACGACGAGTTCAAGCACTGCTGGGACACCTTCGTTGACCACCAGGGCTGCCCATTCCAACCATGGGACGGTCTGGATGAACACAGCCAAGCCTTGTCCGGCAGGCTCCGGGCCATCCTCCAAAACCAGGGGAACTCCGGGAGCGAGACGCCAGGCACCTCCGAGTCGGCCACCCCAGAATCTCTTAAGGACAAGAAGTACTCGATCGGCCTCGACATCGGGACGAACTCAGTTGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTCCTGGGGAACACCGACCGCCATTCCATCAAGAAGAACCTCATCGGCGCTCTCCTGTTCGACAGCGGGGAGACCGCTGAGGCTACGAGGCTCAAGAGAACCGCTAGGCGCCGGTACACGAGAAGGAAGAACAGGATCTGCTACCTCCAAGAGATTTTCTCCAACGAGATGGCCAAGGTTGACGATTCATTCTTCCACCGCCTGGAGGAGTCTTTCCTCGTGGAGGAGGATAAGAAGCACGAGCGGCATCCCATCTTCGGCAACATCGTGGACGAGGTTGCCTACCACGAGAAGTACCCTACGATCTACCATCTGCGGAAGAAGCTCGTGGACTCCACCGATAAGGCGGACCTCAGACTGATCTACCTCGCTCTGGCCCACATGATCAAGTTCCGCGGCCATTTCCTGATCGAGGGGGATCTCAACCCAGACAACAGCGATGTTGACAAGCTGTTCATCCAACTCGTGCAGACCTACAACCAACTCTTCGAGGAGAACCCGATCAACGCCTCTGGCGTGGACGCGAAGGCTATCCTGTCCGCGAGGCTCTCGAAGTCCAGGAGGCTGGAGAACCTGATCGCTCAGCTCCCAGGCGAGAAGAAGAACGGCCTGTTCGGGAACCTCATCGCTCTCAGCCTGGGGCTCACCCCGAACTTCAAGTCGAACTTCGATCTCGCTGAGGACGCCAAGCTGCAACTCTCCAAGGACACCTACGACGATGACCTCGATAACCTCCTGGCCCAGATCGGCGATCAATACGCGGACCTGTTCCTCGCTGCCAAGAACCTGTCGGACGCCATCCTCCTGTCAGATATCCTCCGCGTGAACACCGAGATCACGAAGGCTCCACTCTCTGCCTCCATGATCAAGCGCTACGACGAGCACCATCAGGATCTGACCCTCCTGAAGGCGCTGGTCCGCCAACAGCTCCCGGAGAAGTACAAGGAGATTTTCTTCGATCAGTCGAAGAACGGCTACGCTGGGTACATCGACGGCGGGGCCTCACAAGAGGAGTTCTACAAGTTCATCAAGCCAATCCTGGAGAAGATGGACGGCACGGAGGAGCTCCTGGTGAAGCTCAACAGGGAGGACCTCCTGCGGAAGCAGAGAACCTTCGATAACGGCAGCATCCCCCACCAAATCCATCTCGGGGAGCTGCACGCCATCCTGAGAAGGCAAGAGGACTTCTACCCTTTCCTCAAGGATAACCGGGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCATACTACGTCGGCCCTCTCGCGCGGGGGAACTCAAGATTCGCTTGGATGACCCGCAAGTCTGAGGAGACCATCACGCCGTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCTAGCGCTCAGTCGTTCATCGAGAGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTCCCTAAGCACTCGCTCCTGTACGAGTACTTCACCGTCTACAACGAGCTCACGAAGGTGAAGTACGTCACCGAGGGCATGCGCAAGCCAGCGTTCCTGTCCGGGG
AGCAGAAGAAGGCTATCGTGGACCTCCTGTTCAAGACCAACCGGAAGGTCACGGTTAAGCAACTCAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGATTCGGTCGAGATCAGCGGCGTTGAGGACCGCTTCAACGCCAGCCTCGGGACCTACCACGATCTCCTGAAGATCATCAAGGATAAGGACTTCCTGGACAACGAGGAGAACGAGGATATCCTGGAGGACATCGTGCTGACCCTCACGCTGTTCGAGGACAGGGAGATGATCGAGGAGCGCCTGAAGACGTACGCCCATCTCTTCGATGACAAGGTCATGAAGCAACTCAAGCGCCGGAGATACACCGGCTGGGGGAGGCTGTCCCGCAAGCTCATCAACGGCATCCGGGACAAGCAGTCCGGGAAGACCATCCTCGACTTCCTCAAGAGCGATGGCTTCGCCAACAGGAACTTCATGCAACTGATCCACGATGACAGCCTCACCTTCAAGGAGGATATCCAAAAGGCTCAAGTGAGCGGCCAGGGGGACTCGCTGCACGAGCATATCGCGAACCTCGCTGGCTCCCCCGCGATCAAGAAGGGCATCCTCCAGACCGTGAAGGTTGTGGACGAGCTCGTGAAGGTCATGGGCCGGCACAAGCCTGAGAACATCGTCATCGAGATGGCCAGAGAGAACCAAACCACGCAGAAGGGGCAAAAGAACTCTAGGGAGCGCATGAAGCGCATCGAGGAGGGCATCAAGGAGCTGGGGTCCCAAATCCTCAAGGAGCACCCAGTGGAGAACACCCAACTGCAGAACGAGAAGCTCTACCTGTACTACCTCCAGAACGGCAGGGATATGTACGTGGACCAAGAGCTGGATATCAACCGCCTCAGCGATTACGACGTCGATCATATCGTTCCCCAGTCTTTCCTGAAGGATGACTCCATCGACAACAAGGTCCTCACCAGGTCGGACAAGAACCGCGGCAAGTCAGATAACGTTCCATCTGAGGAGGTCGTTAAGAAGATGAAGAACTACTGGAGGCAGCTCCTGAACGCCAAGCTGATCACGCAAAGGAAGTTCGACAACCTCACCAAGGCTGAGAGAGGCGGGCTCTCAGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTCGAGACCAGACAAATCACGAAGCACGTTGCGCAAATCCTCGACTCTCGGATGAACACGAAGTACGATGAGAACGACAAGCTGATCAGGGAGGTTAAGGTGATCACCCTGAAGTCTAAGCTCGTCTCCGACTTCAGGAAGGATTTCCAGTTCTACAAGGTTCGCGAGATCAACAACTACCACCATGCCCATGACGCTTACCTCAACGCTGTGGTCGGCACCGCTCTGATCAAGAAGTACCCAAAGCTGGAGTCCGAGTTCGTGTACGGGGACTACAAGGTTTACGATGTGCGCAAGATGATCGCCAAGTCGGAGCAAGAGATCGGCAAGGCTACCGCCAAGTACTTCTTCTACTCAAACATCATGAACTTCTTCAAGACCGAGATCACGCTGGCCAACGGCGAGATCCGGAAGAGACCGCTCATCGAGACCAACGGCGAGACGGGGGAGATCGTGTGGGACAAGGGCAGGGATTTCGCGACCGTCCGCAAGGTTCTCTCCATGCCCCAGGTGAACATCGTCAAGAAGACCGAGGTCCAAACGGGCGGGTTCTCAAAGGAGTCTATCCTGCCTAAGCGGAACAGCGACAAGCTCATCGCCAGAAAGAAGGACTGGGACCCAAAGAAGTACGGCGGGTTCGACAGCCCTACCGTGGCCTACTCGGTCCTGGTTGTGGCGAAGGTTGAGAAGGGCAAGTCCAAGAAGCTCAAGAGCGTGAAGGAGCTCCTGGGGATCACCATCATGGAGAGGTCCAGCTTCGAGAAGAACCCAATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTCCCGAAGTACTCTCTCTTCGAGCTGGAGAACGGCAGGAAGAGAATGCTGGCTTCCGCTGGCGAGCTCCAGAAGGGGAACGAGCTCGCGCTGCCAAGCAAGTACGTGAACTTCCTCTACCTGGCTTCCCACTACGAGAAGCTCAAGGGCAGCCCGGAGGACAACGAGCAAAAGCAGCTGTTCGTCGAGCAGCACAAGCATTACCTCGACGAGATCATCGAGCAAATCTCCGAGTTCAGCAAGCGCGTGATCCTCGCCGACGCGAACCTGGATAAGGTCCTCTCCGCCTACAACAAGCACCGGGACAAGCCCATCAGAGAGCAAGCGGAGAACATCATCCATCTCTTCACCCTGACGAACCTCGGCGCTCCTGCTGCTTTCAAGTACTTCGACACCACGATCGATCGGAAGAGATACACCTCCACGAAGGAGGTCCTGGACGCGACCCTCATCCACCAGTCGATCACCGGCCTGTACGAGACGAGGATCGACCTCTCACAACTCGGCGGGGATaagagacccgcagcaaccaagaaggcagggcaagcaaagaagaagaagACGCGTGACTCCGGCGGCAGCGCCAACGAGCTGACCTGGCACGACGTGCTCGCCGAGGAGAAGCAACAGCCATACTTCCTGAACACCCTCCAAACCGTGGCTTCCGAGAGGCAGAGCGGCGTCACCATCTACCCACCACAAAAGGACGTGTTCAACGCCTTCCGCTTCACCGAGCTGGGCGACGTCAAGGTGGTCATCCTCGGCCAAGACCCATACCACGGCCCAGGCCAGGCTCACGGCCTCGCCTTCTCCGTCAGGCCAGGCATCGCCATCCCACCAAGCCTCCTGAACATGTACAAGGAGCTGGAGAACACCATCCCAGGCTTCACCAGGCC
AAACCACGGCTACCTCGAGTCCTGGGCTAGGCAAGGCGTGCTCCTGCTCAACACCGTGCTGACCGTCAGGGCTGGCCAGGCTCACTCCCACGCCAGCCTGGGCTGGGAGACGTTCACCGACAAGGTCATCTCCCTCATCAACCAGCACAGGGAGGGCGTGGTGTTCCTCCTCTGGGGCAGCCACGCCCAAAAGAAGGGCGCCATCATCGACAAGCAGAGGCACCACGTGCTGAAGGCCCCACACCCGTCCCCACTCTCCGCTCACAGGGGCTTCTTCGGCTGCAACCACTTCGTGCTGGCCAACCAATGGCTCGAGCAGAGGGGCGAGACGCCAATCGACTGGATGCCAGTCCTCCCAGCCGAGAGCGAGccaaagaagaagcggaaggtgTCGGCCGGCTCCGGCGCTACGAACTTCTCCCTCCTGAAGCAGGCTGGCGACGTGGAGGAGAACCCAGGCCCGCCGGAGGGCCCAGAGATCAGGCGCGCTGCTGACAACCTCGAGGCTGCCATCAAGGGCAAGCCACTGACCGACGTGTGGTTCGCCTTCCCACAACTCAAGCCATACCAATCCCAGCTGATCGGCCAGCACGTGACCCACGTGGAGACGAGGGGCAAGGCCCTCCTGACCCACTTCTCCAACGACCTGACCCTCTACAGCCACAACCAACTCTACGGCGTGTGGCGCGTGGTGGACACCGGCGAGGAGCCACAAACCACCCGCGTGCTGAGGGTCAAGCTCCAGACCGCCGACAAGACCATCCTCCTGTACTCCGCCAGCGACATCGAGATGCTGACCCCAGAGCAACTCACCACCCACCCATTCCTCCAGCGCGTGGGCCCAGACGTCCTGGACCCAAACCTCACCCCAGAGGTGGTCAAGGAGAGGCTCCTGAGCCCAAGGTTCCGCAACAGGCAATTCGCCGGCCTCCTGCTGGACCAGGCCTTCCTGGCTGGCCTCGGCAACTACCTGCGCGTGGAGATCCTCTGGCAAGTCGGCCTGACCGGCAACCACAAGGCCAAGGACCTCAACGCTGCTCAGCTGGACGCCCTCGCTCACGCCCTGCTCGAGATCCCACGCTTCTCCTACGCCACCAGGGGCCAAGTGGACGAGAACAAGCACCACGGCGCCCTCTTCCGCTTCAAGGTGTTCCACCGCGACGGCGAGCCATGCGAGAGGTGCGGCAGCATCATCGAAAAGACCACCCTGTCCAGCAGGCCATTCTACTGGTGCCCAGGCTGCCAGCACccaaagaagaagcggaaggtgTAG Supplementary Sequence 2: eAFID-3 (A3Bctd-Cas9-UDG-P2A-AP) ATGGAGATCCTCAGGTACCTCATGGACCCAGACACCTTCACCTTCAACTTCAACAACGACCCACTCGTGCTCAGGAGGAGGCAAACGTACCTCTGCTACGAGGTGGAGAGGCTCGATAATGGCACCTGGGTGCTCATGGACCAACACATGGGCTTCCTCTGCAACGAGGCCAAGAATCTCCTCTGCGGCTTCTATGGCAGGCATGCCGAACTCAGGTTTCTCGACCTCGTGCCATCCCTTCAACTCGACCCAGCCCAAATCTACAGGGTGACCTGGTTCATCTCCTGGTCCCCATGTTTTTCCTGGGGCTGTGCCGGAGAAGTGAGGGCCTTTCTCCAAGAGAACACCCACGTGAGGCTCAGGATTTTTGCCGCCAGGATCTACGATTACGACCCACTCTACAAGGAGGCCCTCCAAATGCTTAGGGATGCCGGAGCACAAGTGTCCATCATGACCTACGACGAGTTCGAGTACTGCTGGGACACCTTCGTCTACAGGCAAGGCTGCCCATTCCAACCATGGGACGGCCTCGAAGAACATTCCCAAGCCCTCTCAGGAAGGCTTAGGGCTATCCTCCAAAACCAAGGCAACTCCGGGAGCGAGACGCCAGGCACCTCCGAGTCGGCCACCCCAGAATCTCTTAAGGACAAGAAGTACTCGATCGGCCTCGACATCGGGACGAACTCAGTTGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTCCTGGGGAACACCGACCGCCATTCCATCAAGAAGAACCTCATCGGCGCTCTCCTGTTCGACAGCGGGGAGACCGCTGAGGCTACGAGGCTCAAGAGAACCGCTAGGCGCCGGTACACGAGAAGGAAGAACAGGATCTGCTACCTCCAAGAGATTTTCTCCAACGAGATGGCCAAGGTTGACGATTCATTCTTCCACCGCCTGGAGGAGTCTTTCCTCGTGGAGGAGGATAAGAAGCACGAGCGGCATCCCATCTTCGGCAACATCGTGGACGAGGTTGCCTACCACGAGAAGTACCCTACGATCTACCATCTGCGGAAGAAGCTCGTGGACTCCACCGATAAGGCGGACCTCAGACTGATCTACCTCGCTCTGGCCCACATGATCAAGTTCCGCGGCCATTTCCTGATCGAGGGGGATCTCAACCCAGACAACAGCGATGTTGACAAGCTGTTCATCCAACTCGTGCAGACCTACAACCAACTCTTCGAGGAGAACCCGATCAACGCCTCTGGCGTGGACGCGAAGGCTATCCTGTCCGCGAGGCTCTCGAAGTCCAGGAGGCTGGAGAACCTGATCGCTCAGCTCCCAGGCGAGAAGAAGAACGGCCTGTTCGGGAACCTCATCGCTCTCAGCCTGGGGCTCACCCCGAACTTCAAGTCGAACTTCGATCTCGCTGAGGACGCCAA
GCTGCAACTCTCCAAGGACACCTACGACGATGACCTCGATAACCTCCTGGCCCAGATCGGCGATCAATACGCGGACCTGTTCCTCGCTGCCAAGAACCTGTCGGACGCCATCCTCCTGTCAGATATCCTCCGCGTGAACACCGAGATCACGAAGGCTCCACTCTCTGCCTCCATGATCAAGCGCTACGACGAGCACCATCAGGATCTGACCCTCCTGAAGGCGCTGGTCCGCCAACAGCTCCCGGAGAAGTACAAGGAGATTTTCTTCGATCAGTCGAAGAACGGCTACGCTGGGTACATCGACGGCGGGGCCTCACAAGAGGAGTTCTACAAGTTCATCAAGCCAATCCTGGAGAAGATGGACGGCACGGAGGAGCTCCTGGTGAAGCTCAACAGGGAGGACCTCCTGCGGAAGCAGAGAACCTTCGATAACGGCAGCATCCCCCACCAAATCCATCTCGGGGAGCTGCACGCCATCCTGAGAAGGCAAGAGGACTTCTACCCTTTCCTCAAGGATAACCGGGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCATACTACGTCGGCCCTCTCGCGCGGGGGAACTCAAGATTCGCTTGGATGACCCGCAAGTCTGAGGAGACCATCACGCCGTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCTAGCGCTCAGTCGTTCATCGAGAGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTCCCTAAGCACTCGCTCCTGTACGAGTACTTCACCGTCTACAACGAGCTCACGAAGGTGAAGTACGTCACCGAGGGCATGCGCAAGCCAGCGTTCCTGTCCGGGGAGCAGAAGAAGGCTATCGTGGACCTCCTGTTCAAGACCAACCGGAAGGTCACGGTTAAGCAACTCAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGATTCGGTCGAGATCAGCGGCGTTGAGGACCGCTTCAACGCCAGCCTCGGGACCTACCACGATCTCCTGAAGATCATCAAGGATAAGGACTTCCTGGACAACGAGGAGAACGAGGATATCCTGGAGGACATCGTGCTGACCCTCACGCTGTTCGAGGACAGGGAGATGATCGAGGAGCGCCTGAAGACGTACGCCCATCTCTTCGATGACAAGGTCATGAAGCAACTCAAGCGCCGGAGATACACCGGCTGGGGGAGGCTGTCCCGCAAGCTCATCAACGGCATCCGGGACAAGCAGTCCGGGAAGACCATCCTCGACTTCCTCAAGAGCGATGGCTTCGCCAACAGGAACTTCATGCAACTGATCCACGATGACAGCCTCACCTTCAAGGAGGATATCCAAAAGGCTCAAGTGAGCGGCCAGGGGGACTCGCTGCACGAGCATATCGCGAACCTCGCTGGCTCCCCCGCGATCAAGAAGGGCATCCTCCAGACCGTGAAGGTTGTGGACGAGCTCGTGAAGGTCATGGGCCGGCACAAGCCTGAGAACATCGTCATCGAGATGGCCAGAGAGAACCAAACCACGCAGAAGGGGCAAAAGAACTCTAGGGAGCGCATGAAGCGCATCGAGGAGGGCATCAAGGAGCTGGGGTCCCAAATCCTCAAGGAGCACCCAGTGGAGAACACCCAACTGCAGAACGAGAAGCTCTACCTGTACTACCTCCAGAACGGCAGGGATATGTACGTGGACCAAGAGCTGGATATCAACCGCCTCAGCGATTACGACGTCGATCATATCGTTCCCCAGTCTTTCCTGAAGGATGACTCCATCGACAACAAGGTCCTCACCAGGTCGGACAAGAACCGCGGCAAGTCAGATAACGTTCCATCTGAGGAGGTCGTTAAGAAGATGAAGAACTACTGGAGGCAGCTCCTGAACGCCAAGCTGATCACGCAAAGGAAGTTCGACAACCTCACCAAGGCTGAGAGAGGCGGGCTCTCAGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTCGAGACCAGACAAATCACGAAGCACGTTGCGCAAATCCTCGACTCTCGGATGAACACGAAGTACGATGAGAACGACAAGCTGATCAGGGAGGTTAAGGTGATCACCCTGAAGTCTAAGCTCGTCTCCGACTTCAGGAAGGATTTCCAGTTCTACAAGGTTCGCGAGATCAACAACTACCACCATGCCCATGACGCTTACCTCAACGCTGTGGTCGGCACCGCTCTGATCAAGAAGTACCCAAAGCTGGAGTCCGAGTTCGTGTACGGGGACTACAAGGTTTACGATGTGCGCAAGATGATCGCCAAGTCGGAGCAAGAGATCGGCAAGGCTACCGCCAAGTACTTCTTCTACTCAAACATCATGAACTTCTTCAAGACCGAGATCACGCTGGCCAACGGCGAGATCCGGAAGAGACCGCTCATCGAGACCAACGGCGAGACGGGGGAGATCGTGTGGGACAAGGGCAGGGATTTCGCGACCGTCCGCAAGGTTCTCTCCATGCCCCAGGTGAACATCGTCAAGAAGACCGAGGTCCAAACGGGCGGGTTCTCAAAGGAGTCTATCCTGCCTAAGCGGAACAGCGACAAGCTCATCGCCAGAAAGAAGGACTGGGACCCAAAGAAGTACGGCGGGTTCGACAGCCCTACCGTGGCCTACTCGGTCCTGGTTGTGGCGAAGGTTGAGAAGGGCAAGTCCAAGAAGCTCAAGAGCGTGAAGGAGCTCCTGGGGATCACCATCATGGAGAGGTCCAGCTTCGAGAAGAACCCAATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTCCCGAAGTACTCTCTCTTCGAGCTGGAGAACGGCAGGAAGAGAATGCTGGCTTCC
GCTGGCGAGCTCCAGAAGGGGAACGAGCTCGCGCTGCCAAGCAAGTACGTGAACTTCCTCTACCTGGCTTCCCACTACGAGAAGCTCAAGGGCAGCCCGGAGGACAACGAGCAAAAGCAGCTGTTCGTCGAGCAGCACAAGCATTACCTCGACGAGATCATCGAGCAAATCTCCGAGTTCAGCAAGCGCGTGATCCTCGCCGACGCGAACCTGGATAAGGTCCTCTCCGCCTACAACAAGCACCGGGACAAGCCCATCAGAGAGCAAGCGGAGAACATCATCCATCTCTTCACCCTGACGAACCTCGGCGCTCCTGCTGCTTTCAAGTACTTCGACACCACGATCGATCGGAAGAGATACACCTCCACGAAGGAGGTCCTGGACGCGACCCTCATCCACCAGTCGATCACCGGCCTGTACGAGACGAGGATCGACCTCTCACAACTCGGCGGGGATaagagacccgcagcaaccaagaaggcagggcaagcaaagaagaagaagACGCGTGACTCCGGCGGCAGCGCCAACGAGCTGACCTGGCACGACGTGCTCGCCGAGGAGAAGCAACAGCCATACTTCCTGAACACCCTCCAAACCGTGGCTTCCGAGAGGCAGAGCGGCGTCACCATCTACCCACCACAAAAGGACGTGTTCAACGCCTTCCGCTTCACCGAGCTGGGCGACGTCAAGGTGGTCATCCTCGGCCAAGACCCATACCACGGCCCAGGCCAGGCTCACGGCCTCGCCTTCTCCGTCAGGCCAGGCATCGCCATCCCACCAAGCCTCCTGAACATGTACAAGGAGCTGGAGAACACCATCCCAGGCTTCACCAGGCCAAACCACGGCTACCTCGAGTCCTGGGCTAGGCAAGGCGTGCTCCTGCTCAACACCGTGCTGACCGTCAGGGCTGGCCAGGCTCACTCCCACGCCAGCCTGGGCTGGGAGACGTTCACCGACAAGGTCATCTCCCTCATCAACCAGCACAGGGAGGGCGTGGTGTTCCTCCTCTGGGGCAGCCACGCCCAAAAGAAGGGCGCCATCATCGACAAGCAGAGGCACCACGTGCTGAAGGCCCCACACCCGTCCCCACTCTCCGCTCACAGGGGCTTCTTCGGCTGCAACCACTTCGTGCTGGCCAACCAATGGCTCGAGCAGAGGGGCGAGACGCCAATCGACTGGATGCCAGTCCTCCCAGCCGAGAGCGAGccaaagaagaagcggaaggtgTCGGCCGGCTCCGGCGCTACGAACTTCTCCCTCCTGAAGCAGGCTGGCGACGTGGAGGAGAACCCAGGCCCGCCGGAGGGCCCAGAGATCAGGCGCGCTGCTGACAACCTCGAGGCTGCCATCAAGGGCAAGCCACTGACCGACGTGTGGTTCGCCTTCCCACAACTCAAGCCATACCAATCCCAGCTGATCGGCCAGCACGTGACCCACGTGGAGACGAGGGGCAAGGCCCTCCTGACCCACTTCTCCAACGACCTGACCCTCTACAGCCACAACCAACTCTACGGCGTGTGGCGCGTGGTGGACACCGGCGAGGAGCCACAAACCACCCGCGTGCTGAGGGTCAAGCTCCAGACCGCCGACAAGACCATCCTCCTGTACTCCGCCAGCGACATCGAGATGCTGACCCCAGAGCAACTCACCACCCACCCATTCCTCCAGCGCGTGGGCCCAGACGTCCTGGACCCAAACCTCACCCCAGAGGTGGTCAAGGAGAGGCTCCTGAGCCCAAGGTTCCGCAACAGGCAATTCGCCGGCCTCCTGCTGGACCAGGCCTTCCTGGCTGGCCTCGGCAACTACCTGCGCGTGGAGATCCTCTGGCAAGTCGGCCTGACCGGCAACCACAAGGCCAAGGACCTCAACGCTGCTCAGCTGGACGCCCTCGCTCACGCCCTGCTCGAGATCCCACGCTTCTCCTACGCCACCAGGGGCCAAGTGGACGAGAACAAGCACCACGGCGCCCTCTTCCGCTTCAAGGTGTTCCACCGCGACGGCGAGCCATGCGAGAGGTGCGGCAGCATCATCGAAAAGACCACCCTGTCCAGCAGGCCATTCTACTGGTGCCCAGGCTGCCAGCACccaaagaagaagcggaaggtgTAG
