CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors
Item Type Article
Authors Butt, Haroon;Eid, Ayman;Momin, Afaque Ahmad Imtiyaz;Bazin, Jeremie;Crespi, Martin;Arold, Stefan T.;Mahfouz, Magdy M.
Citation Butt H, Eid A, Momin AA, Bazin J, Crespi M, et al. (2019) CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors. Genome Biology 20. Available: http://
dx.doi.org/10.1186/s13059-019-1680-9.
Eprint version Publisher's Version/PDF
DOI 10.1186/s13059-019-1680-9
Publisher Springer Nature
Journal Genome Biology
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Download date 2023-10-31 16:35:53
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Link to Item http://hdl.handle.net/10754/652834
1 Additional file 2
CRISPR-directed evolution of the spliceosome for resistance to splicing inhibitors
Haroon Butt
1, Ayman Eid
1, Afaque A. Momin
2, Jeremie Bazin
3, Martin Crespi
3, Stefan T. Arold
2, and Magdy M. Mahfouz
1,*1
Laboratory for Genome Engineering and Synthetic Biology, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia,
2Division of Biological and Environmental Sciences and Engineering, Computational Bioscience Research Center, KAUST, Thuwal, Saudi Arabia, and
3CNRS, INRA, Institute of Plant Sciences Paris-Saclay IPS2, Univ Paris Sud, Univ Evry, Univ Paris-Diderot, Sorbonne Paris-Cite, Universite Paris-Saclay, Orsay, France.
*
Correspondence to Magdy Mahfouz ([email protected])
Additional File 2:
Table S1: Sequences of sgRNAs with PAM targeting SF3B1
Table S2: List of primers annealed to synthesis sgRNAs with BsaI compatible overhangs Table S3: List of primers used for genotyping
Table S4: CDE-platform for SF3B1 resistance to splicing inhibitor Table S5: List of primers used for Intron retention analysis
Table S1: Sequences of sgRNAs with PAM targeting SF3B1
2 sgRNA 5´-sgRNA sequence-3´ (PAM is underlined)
1
GGAGCTCGCGCGGGCGCAGGAGG2
GGAGCGCAAGAAGATGGAGGAGG3
CCGCCGGCGCGCCCATGGCCGTC4
CCTTCGACACCGATCTCTACGGC5
CCGACCCCAACCGCTTCGCCGGC6
CCTCCATCCCCGCCTCCGAGGAC7
CTCCGAGGACGACGCGCCCGAGG8
CCGAGGCGGCCGTCAACCCCGCC9
CCCGCCGCCTCGCCTCCTACACC10
CCGGCCACGCCGTCGCCGCCGCC11
CGACATCCCCCGCGCCGCCGAGG12
CCAAGAAGTCGCAGCGCATCATC13
CCGCGAGGATGACTATCGCCGCC14
GCTGGCCCGCATCATCTCGCCGG15
CCGGAGCGCCATGATCCCTTCGC16
CCACCCCGGACCCTTCCGTGCGG17
CCGATGCCATGCGCGAGAATGAC18
CCTGCAGAAGCAGAAGGAGCAGC19
TGACATAGCTCAGAAGAAGAAGG20
GGAGAAGGCCAAGGAGAAGAAGG21
GCCGAAGCGCCGCAATAGATGGG22
CAATAGATGGGATCAGTCGCAGG23
GGTGATGCTTCTGCTGCTGCTGG24
GGCGAAGACCTCCTCCGATTGGG25
GATGCTCCTGATGCAACTCCTGG26
GGGCGCTGGGATGCCACCCCTGG27
CCCCTGGTCGTGTCGGGGATGCG28
GTCGGTGAGAAGGAATCGTTGGG29
CCGTCGGTGAGAAGGAATCGTTG30
CCGATGCGGATGCAACACCTGCA31
GCAGCTGGTGGCATTACTCCAGG32
CCTTCTGGAGCTTGGGATGCTAC33
CCTGGTGGGCTTGTCACGCCAAC34
CCAACACCCAAGAAGCAGAGATC35
CCTGCAAGTATGGGTAGTGCTAC36
CCTGGAGGCACTGGTGCTGCGAC37
CCTGCAGGTTACACTCCTGGTCC38
CCATTTGGTGGTGACAACCTTGC39
CCAACACCCGGCCAGATTGCTTC40
CAGAGCAGTACCAGCTCTTGCGG41
GAACAGGCCTCTTACGGATGAGG42
GCTTGATACTATGTTCCCGCAGG43
CCGCAGGAAGGGTACAAGATTCT44
CCAGCTTCATACCAACCCATACG45
CCAGCAAGAAAGCTGCTCGCTAC46
ACCATTGTATGCTATTCCTGAGG47
GCTATTCCTGAGGAGAATCGTGG48
GTGCCCAAGGAGTTGCCTGGTGG49
GTTGCCCCTCATGAAGCCAGAGG50
CCAGTACTTTGGAACATTGCTGA51
GCTATCTCCAGAAGAGCAGAAGG52
GATCATGAAGCTCCTGCTCAAGG53
CCACAGCGGAAGACAGCACTTCG3 54
GCTCATGCAGCCAACACTTGAGG55
TGACAGGGTACTGTATAAGCTGG56
CCGTCCGTTTGTGCATAAGATTC57
GACTACTATGCTCGTGTTGAGGG58
CCGGGAGATTATCTCAAATCTTA59
TCAAATCTTAGCAAAGCTGCTGG60
CCGCCATGAGACCAGATATTGAT61
CACTGCTAGAGCATTTAGCGTGG62
TAGCGTGGTGGCTTCTGCTTTGG63
TCCTGGCAAGCTCGGCACACTGG64
CCTGGCAAGCTCGGCACACTGGT65
CAGCAGATTGCTATTCTTATGGG66
CCTAGTTGAGATCATTGAGCATG67
GTGACGAGAACCAGAAAGTGCGG68
CCAGAAAGTGCGGACAATCACTG69
AAGGGTATCAGATCTCACCGTGG70
CCGTGGAAAGGTTCTTGCTGCCT71
GCTGCCTTCTTGAAAGCTATTGG72
CCTCTTATGGATGCGCTGTATGC73
CCTGATTAGGGAGTTCCAGTCAC74
GATGAAGAAGATTGTCCTGAAGG75
GTGTAGAGGCTGATTATATCCGG76
GGAGAATGGCTCTGGACCGCAGG77
GTAGAGATGGCAAATAAGGTTGG78
TATTGTTGGGAGGATTGTTGAGG79
GCCTTACAGGAGAATGGTGATGG80
GGTGATGGAAACAATTGAGAAGG81
TTGGAGGAGCTGCTTATTGATGG82
CCTTACCTTCCTCAGATTTGTGG83
AAGCTGCTGATCTGATCTCGAGG84
GTGTCAGGAGGAGCAGCTTATGG85
GAAGAGTACCCTGAGGTGTTGGG86
CCGCCAATCAAGGATCTTCTTCC87
GAATAGGCATGAGAAGGTCCAGG88
GAGAACTGCATTGATCTAGTTGG89
GTTGGTAGGATTGCTGATCGTGG90
TGCCTGCTAGGGAGTGGATGAGG91
AATGTTGAAGGCTCACAAGAAGG92 (HR-target)
ATGTTGAAGGCTCACAAGAAGGG93
AGGCTCACAAGAAGGGTATTAGA94
CTCACAAGAAGGGTATTAGAAGA95
GGTTATATTGCGAAGGCTATTGG96
GTTGAATAACTTGAAGGTGCAGG97
CCGTGTCTGCACAACTGTCGCAA98
CCTGATGAATGAGTACCGAGTCC99
GTATATTGGTGAGATGGGCAAGG100
CCTTGCTTGAAGATGCTCTAATG101
GCTGTGAAGCACATGGCTCTTGG102
TGTTGCTGGCTTGGGCTGTGAGG103
GCTGTCATGGAGGCGATTGAGGG104
GAGGGGATGAGAGTTGCTCTTGG105
CCAGCTGTGATCTTGAATTACTG106
CCATCCAGCAAGGAAAGTACGTG107
TCCTGCACTGGATGACGATGGGG4 108
TATCTACAGCCGTCCAGAGCTGG109
GATGAGACTCCTGCAAGTATGGG110
CCAGAAGAGCAGAAGGAGAGGAA111
AAAGCAAGGGAATTTGGTGCTGG112
CCATTGTTCAACAAGATTCTACC113
CCAGATATTGATAACATTGATGA114
ATAACATTGATGAGTATGTGAGA115
CCACTGCTAGAGCATTTAGCGTG116
CCAGTCACCAGATGAAGAGATGA117
CCAGATGAAGAGATGAAGAAGAT118
CCGGCAAACTGCTGCCTCTGCTG119
CACATTTGGTTATATTGCGAAGGTable S2: List of primers annealed to synthesis sgRNAs with BsaI compatible overhangs
Name of the primer Sequence (5' - 3')
F1
GGCAGGAGCTCGCGCGGGCGCAGGF2
GGCAGGAGCGCAAGAAGATGGAGGF3
GGCAGACGGCCATGGGCGCGCCGG5
F4
GGCAGCCGTAGAGATCGGTGTCGAF5
GGCAGCCGGCGAAGCGGTTGGGGTF6
GGCAGTCCTCGGAGGCGGGGATGGF7
GGCACTCCGAGGACGACGCGCCCGF8
GGCAGGCGGGGTTGACGGCCGCCTF9
GGCAGGTGTAGGAGGCGAGGCGGCF10
GGCAGGCGGCGGCGACGGCGTGGCF11
GGCACGACATCCCCCGCGCCGCCGF12
GGCAGATGATGCGCTGCGACTTCTF13
GGCAGGCGGCGATAGTCATCCTCGF14
GGCAGCTGGCCCGCATCATCTCGCF15
GGCAGCGAAGGGATCATGGCGCTCF16
GGCACCACCCCGGACCCTTCCGTGF17
GGCAGTCATTCTCGCGCATGGCATF18
GGCAGCTGCTCCTTCTGCTTCTGCF19
GGCATGACATAGCTCAGAAGAAGAF20
GGCAGGAGAAGGCCAAGGAGAAGAF21
GGCAGCCGAAGCGCCGCAATAGATF22
GGCACAATAGATGGGATCAGTCGCF23
GGCAGGTGATGCTTCTGCTGCTGCF24
GGCAGGCGAAGACCTCCTCCGATTF25
GGCAGATGCTCCTGATGCAACTCCF26
GGCAGGGCGCTGGGATGCCACCCCF27
GGCACGCATCCCCGACACGACCAGF28
GGCAGTCGGTGAGAAGGAATCGTTF29
GGCACAACGATTCCTTCTCACCGAF30
GGCATGCAGGTGTTGCATCCGCATF31
GGCAGCAGCTGGTGGCATTACTCCF32
GGCAGTAGCATCCCAAGCTCCAGAF33
GGCAGTTGGCGTGACAAGCCCACCF34
GGCAGATCTCTGCTTCTTGGGTGTF35
GGCAGTAGCACTACCCATACTTGCF36
GGCAGTCGCAGCACCAGTGCCTCCF37
GGCAGGACCAGGAGTGTAACCTGCF38
GGCAGCAAGGTTGTCACCACCAAAF39
GGCAGAAGCAATCTGGCCGGGTGTF40
GGCACAGAGCAGTACCAGCTCTTGF41
GGCAGAACAGGCCTCTTACGGATGF42
GGCAGCTTGATACTATGTTCCCGCF43
GGCAAGAATCTTGTACCCTTCCTGF44
GGCACGTATGGGTTGGTATGAAGCF45
GGCAGTAGCGAGCAGCTTTCTTGCF46
GGCAACCATTGTATGCTATTCCTGF47
GGCAGCTATTCCTGAGGAGAATCG6
F48
GGCAGTGCCCAAGGAGTTGCCTGGF49
GGCAGTTGCCCCTCATGAAGCCAGF50
GGCATCAGCAATGTTCCAAAGTACF51
GGCAGCTATCTCCAGAAGAGCAGAF52
GGCAGATCATGAAGCTCCTGCTCAF53
GGCACGAAGTGCTGTCTTCCGCTGF54
GGCAGCTCATGCAGCCAACACTTGF55
GGCATGACAGGGTACTGTATAAGCF56
GGCAGAATCTTATGCACAAACGGAF57
GGCAGACTACTATGCTCGTGTTGAF58
GGCATAAGATTTGAGATAATCTCCF59
GGCATCAAATCTTAGCAAAGCTGCF60
GGCAATCAATATCTGGTCTCATGGF61
GGCACACTGCTAGAGCATTTAGCGF62
GGCATAGCGTGGTGGCTTCTGCTTF63
GGCATCCTGGCAAGCTCGGCACACF64
GGCAACCAGTGTGCCGAGCTTGCCF65
GGCACAGCAGATTGCTATTCTTATF66
GGCACATGCTCAATGATCTCAACTF67
GGCAGTGACGAGAACCAGAAAGTGF68
GGCACAGTGATTGTCCGCACTTTCF69
GGCAAAGGGTATCAGATCTCACCGF70
GGCAAGGCAGCAAGAACCTTTCCAF71
GGCAGCTGCCTTCTTGAAAGCTATF72
GGCAGCATACAGCGCATCCATAAGF73
GGCAGTGACTGGAACTCCCTAATCF74
GGCAGATGAAGAAGATTGTCCTGAF75
GGCAGTGTAGAGGCTGATTATATCF76
GGCAGGAGAATGGCTCTGGACCGCF77
GGCAGTAGAGATGGCAAATAAGGTF78
GGCATATTGTTGGGAGGATTGTTGF79
GGCAGCCTTACAGGAGAATGGTGAF80
GGCAGGTGATGGAAACAATTGAGAF81
GGCATTGGAGGAGCTGCTTATTGAF82
GGCACCACAAATCTGAGGAAGGTAF83
GGCAAAGCTGCTGATCTGATCTCGF84
GGCAGTGTCAGGAGGAGCAGCTTAF85
GGCAGAAGAGTACCCTGAGGTGTTF86
GGCAGGAAGAAGATCCTTGATTGGF87
GGCAGAATAGGCATGAGAAGGTCCF88
GGCAGAGAACTGCATTGATCTAGTF89
GGCAGTTGGTAGGATTGCTGATCGF90
GGCATGCCTGCTAGGGAGTGGATGF91
GGCAAATGTTGAAGGCTCACAAGA7 F92 (HR-target)
GGCAATGTTGAAGGCTCACAAGAAF93
GGCAAGGCTCACAAGAAGGGTATTF94
GGCACTCACAAGAAGGGTATTAGAF95
GGCAGGTTATATTGCGAAGGCTATF96
GGCAGTTGAATAACTTGAAGGTGCF97
GGCATTGCGACAGTTGTGCAGACAF98
GGCAGGACTCGGTACTCATTCATCF99
GGCAGTATATTGGTGAGATGGGCAF100
GGCACATTAGAGCATCTTCAAGCAF101
GGCAGCTGTGAAGCACATGGCTCTF102
GGCATGTTGCTGGCTTGGGCTGTGF103
GGCAGCTGTCATGGAGGCGATTGAF104
GGCAGAGGGGATGAGAGTTGCTCTF105
GGCACAGTAATTCAAGATCACAGCF106
GGCACACGTACTTTCCTTGCTGGAF107
GGCATCCTGCACTGGATGACGATGF108
GGCATATCTACAGCCGTCCAGAGCF109
GGCAGATGAGACTCCTGCAAGTATF110
GGCATTCCTCTCCTTCTGCTCTTCF111
GGCAAAAGCAAGGGAATTTGGTGCF112
GGCAGGTAGAATCTTGTTGAACAAF113
GGCATCATCAATGTTATCAATATCF114
GGCAATAACATTGATGAGTATGTGF115
GGCACACGCTAAATGCTCTAGCAGF116
GGCATCATCTCTTCATCTGGTGACF117
GGCAATCTTCTTCATCTCTTCATCF118
GGCACAGCAGAGGCAGCAGTTTGCF119
GGCACACATTTGGTTATATTGCGAR1
AAACCCTGCGCCCGCGCGAGCTCCR2
AAACCCTCCATCTTCTTGCGCTCCR3
AAACCCGGCGCGCCCATGGCCGTCR4
AAACTCGACACCGATCTCTACGGCR5
AAACACCCCAACCGCTTCGCCGGCR6
AAACCCATCCCCGCCTCCGAGGACR7
AAACCGGGCGCGTCGTCCTCGGAGR8
AAACAGGCGGCCGTCAACCCCGCCR9
AAACGCCGCCTCGCCTCCTACACCR10
AAACGCCACGCCGTCGCCGCCGCCR11
AAACCGGCGGCGCGGGGGATGTCGR12
AAACAGAAGTCGCAGCGCATCATCR13
AAACCGAGGATGACTATCGCCGCCR14
AAACGCGAGATGATGCGGGCCAGCR15
AAACGAGCGCCATGATCCCTTCGCR16
AAACCACGGAAGGGTCCGGGGTGG8
R17
AAACATGCCATGCGCGAGAATGACR18
AAACGCAGAAGCAGAAGGAGCAGCR19
AAACTCTTCTTCTGAGCTATGTCAR20
AAACTCTTCTCCTTGGCCTTCTCCR21
AAACATCTATTGCGGCGCTTCGGCR22
AAACGCGACTGATCCCATCTATTGR23
AAACGCAGCAGCAGAAGCATCACCR24
AAACAATCGGAGGAGGTCTTCGCCR25
AAACGGAGTTGCATCAGGAGCATCR26
AAACGGGGTGGCATCCCAGCGCCCR27
AAACCTGGTCGTGTCGGGGATGCGR28
AAACAACGATTCCTTCTCACCGACR29
AAACTCGGTGAGAAGGAATCGTTGR30
AAACATGCGGATGCAACACCTGCAR31
AAACGGAGTAATGCCACCAGCTGCR32
AAACTCTGGAGCTTGGGATGCTACR33
AAACGGTGGGCTTGTCACGCCAACR34
AAACACACCCAAGAAGCAGAGATCR35
AAACGCAAGTATGGGTAGTGCTACR36
AAACGGAGGCACTGGTGCTGCGACR37
AAACGCAGGTTACACTCCTGGTCCR38
AAACTTTGGTGGTGACAACCTTGCR39
AAACACACCCGGCCAGATTGCTTCR40
AAACCAAGAGCTGGTACTGCTCTGR41
AAACCATCCGTAAGAGGCCTGTTCR42
AAACGCGGGAACATAGTATCAAGCR43
AAACCAGGAAGGGTACAAGATTCTR44
AAACGCTTCATACCAACCCATACGR45
AAACGCAAGAAAGCTGCTCGCTACR46
AAACCAGGAATAGCATACAATGGTR47
AAACCGATTCTCCTCAGGAATAGCR48
AAACCCAGGCAACTCCTTGGGCACR49
AAACCTGGCTTCATGAGGGGCAACR50
AAACGTACTTTGGAACATTGCTGAR51
AAACTCTGCTCTTCTGGAGATAGCR52
AAACTGAGCAGGAGCTTCATGATCR53
AAACCAGCGGAAGACAGCACTTCGR54
AAACCAAGTGTTGGCTGCATGAGCR55
AAACGCTTATACAGTACCCTGTCAR56
AAACTCCGTTTGTGCATAAGATTCR57
AAACTCAACACGAGCATAGTAGTCR58
AAACGGAGATTATCTCAAATCTTAR59
AAACGCAGCTTTGCTAAGATTTGAR60
AAACCCATGAGACCAGATATTGAT9
R61
AAACCGCTAAATGCTCTAGCAGTGR62
AAACAAGCAGAAGCCACCACGCTAR63
AAACGTGTGCCGAGCTTGCCAGGAR64
AAACGGCAAGCTCGGCACACTGGTR65
AAACATAAGAATAGCAATCTGCTGR66
AAACAGTTGAGATCATTGAGCATGR67
AAACCACTTTCTGGTTCTCGTCACR68
AAACGAAAGTGCGGACAATCACTGR69
AAACCGGTGAGATCTGATACCCTTR70
AAACTGGAAAGGTTCTTGCTGCCTR71
AAACATAGCTTTCAAGAAGGCAGCR72
AAACCTTATGGATGCGCTGTATGCR73
AAACGATTAGGGAGTTCCAGTCACR74
AAACTCAGGACAATCTTCTTCATCR75
AAACGATATAATCAGCCTCTACACR76
AAACGCGGTCCAGAGCCATTCTCCR77
AAACACCTTATTTGCCATCTCTACR78
AAACCAACAATCCTCCCAACAATAR79
AAACTCACCATTCTCCTGTAAGGCR80
AAACTCTCAATTGTTTCCATCACCR81
AAACTCAATAAGCAGCTCCTCCAAR82
AAACTACCTTCCTCAGATTTGTGGR83
AAACCGAGATCAGATCAGCAGCTTR84
AAACTAAGCTGCTCCTCCTGACACR85
AAACAACACCTCAGGGTACTCTTCR86
AAACCCAATCAAGGATCTTCTTCCR87
AAACGGACCTTCTCATGCCTATTCR88
AAACACTAGATCAATGCAGTTCTCR89
AAACCGATCAGCAATCCTACCAACR90
AAACCATCCACTCCCTAGCAGGCAR91
AAACTCTTGTGAGCCTTCAACATTR92 (HR-target)
AAACTTCTTGTGAGCCTTCAACATR93
AAACAATACCCTTCTTGTGAGCCTR94
AAACTCTAATACCCTTCTTGTGAGR95
AAACATAGCCTTCGCAATATAACCR96
AAACGCACCTTCAAGTTATTCAACR97
AAACTGTCTGCACAACTGTCGCAAR98
AAACGATGAATGAGTACCGAGTCCR99
AAACTGCCCATCTCACCAATATACR100
AAACTGCTTGAAGATGCTCTAATGR101
AAACAGAGCCATGTGCTTCACAGCR102
AAACCACAGCCCAAGCCAGCAACAR103
AAACTCAATCGCCTCCATGACAGCR104
AAACAGAGCAACTCTCATCCCCTC10
R105
AAACGCTGTGATCTTGAATTACTGR106
AAACTCCAGCAAGGAAAGTACGTGR107
AAACCATCGTCATCCAGTGCAGGAR108
AAACGCTCTGGACGGCTGTAGATAR109
AAACATACTTGCAGGAGTCTCATCR110
AAACGAAGAGCAGAAGGAGAGGAAR111
AAACGCACCAAATTCCCTTGCTTTR112
AAACTTGTTCAACAAGATTCTACCR113
AAACGATATTGATAACATTGATGAR114
AAACCACATACTCATCAATGTTATR115
AAACCTGCTAGAGCATTTAGCGTGR116
AAACGTCACCAGATGAAGAGATGAR117
AAACGATGAAGAGATGAAGAAGATR118
AAACGCAAACTGCTGCCTCTGCTGR119
AAACTCGCAATATAACCAAATGTGTable S3: List of primers used for genotyping
Name of the primer Sequence (5' - 3')
F1
CTAACCGCCTCTCCCCCCGCCF2
AAGAAGATGGAGGAGGCCCTTF3
CTTCCGTGCGGACCTATGCCF4
AAGGAGAAGAAGGCCGTGCCCF5
ACCGTCGGTGAGAAGGAATCGT11
F6
AGAAAGCTGCTCGCTACACCAF7
GCGGAAGACAGCACTTCGACAAF8
CAAAGCTGCTGGTCTTGCTACCF9
CCACATCTCAAGTCCCTAGTTF10
GATGGAAACAATTGAGAAGGTTF11
ACAAGCTGCTGATCTGATCTCGF12
GCAAAACCGTGTCTGCACAF13
TTGCTTAACTATGTCTGGCCAR1
CAACGGCAACAAAGTAAACATTR2
CCACCGAGTTCAAGCTTCACAGR3
CATAGAATTCTAACATATCAATGTAR4
CAATAATATATGCCACAAAATTTAAGCR5
TCATCAGGGCAGGCAGAACCR6
ATAAGCTGCTCCTCCTGACACR7
TGAAGCACCCAAATTAGCCACAR8
GTCTCATGGCGGCAATCATGR9
GGTAATCCTCTGGCTTCATGAGR10
ACGATTCCTTCTCACCGACGGTR11
CTGCCACTGGCTGCTGCTCGR12
CTTCCTTCTTCTTCTGAGCTATGT-DNA-F
CACAGGAAACAGCTATGACT-DNA-R
CGCGCTAAAAACGGACTAG12 Table S4: CDE-platform for SF3B1 resistance to splicing inhibitor
*Please note that all false positives exhibit no SF3B1 editing and their seed progeny is sensitive to GEX1A
**Please note that one set of transformation was conducted without GEX1A selection for targeted mutagenesis of SF3B1 and no functional knock out mutant could be recovered from these (except one in-frame mutant SGS1).
CRISPR-directed evolution of
SF3B1 via targeted sgRNA library Directed evolution of SF3B1 via domain-specific sgRNAs
No. of transformation 2 1 (One for each sgRNA)
No. of sgRNAs 119 (in an sgRNA libarary
transformed to Agrobacterium)
3
sgRNA-92 sgRNA-119 PTG
No. of calli transformed ~15,000 1000 1000 1000
No. of shoots/plants recovered
on GEX1A supplemented media 21 seedlings from 0.4µM 8 seedlings
from 0.4µM 4 seedlings
from 0.6µM No seedlings 5 seedlings from 0.4µM No. of plants showing SF3B1
editing
SGR1 SGR2 SGR3 SGR3 SGR5 SGR4
___
SGR6
2 2 3 3 2 2 2
Heritability of GEX1A
resistance in seed progeny (T1)
SGR1 SGR2 SGR3 SGR3 SGR5 SGR4 ___ SGR6
2 2 3 3 2 2 2
13 Table S5: List of primers used for Intron retention analysis
Name of the primer Sequence (5' - 3') 03g55590_F
GCTACTCCAAAGCAAATCAGGGAG03g55590_R
CGGACATAGCCTTCCGACTTG04g37790_F
GTCTTCTAATTCGGTTCCCTGACACG04g37790_R
GGGGACGGCTTGGAATCCTTG04g57440_F
GCTAAGGTTGTGAGCATCGATGG04g57440_R
CCAAGAGTTGCGGACAATCCAG07g08460_F
GGGAGGAGGGCTGTATGTGAAG07g08460_R
CTGGCTTCAGAGCCCTTCATG07g35350_F
CGGACTGCAGTGCTATCCAACAG07g35350_R
CATTGAATGCATAGGTCGCATGCG07g48510_F
GGAGGTGCTGTTCTTGCAAGTG07g48510_R
TTGGGACAAGGTCTTGGTTTGG08g36910_F
GAGATCGATCAGTAGTGGTTAGCAG08g36910_R
GCGTCCAGGTCGTAGAGACG08g44390_F
GAGAAGCTCTACGCAGTCTTGG08g44390_R
GGACAATGAAGGGAATGACCATTG10g31850_F
GCATGGGGAAATACTTCTGCG10g31850_R
TCGAAGCAGACAGGACAGTTG