SUPPORTING INFORMATION FOR PUBLICATION: FUNCTIONAL ...
SUPPORTING INFORMATION FOR PUBLICATION: FUNCTIONAL RECONSTITUTION OF A FUNGAL NATURAL PRODUCT GENE CLUSTER BY ADVANCED GENOME EDITING Jakob Weber [1,2], Vito Valiante [3], Christina S. Nødvig [4], Derek J. Mattern [1,2], Rebecca A. Slotkowski [1], Uffe H. Mortensen [4] and Axel A. Brakhage [1,2] * [1] Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI), Jena, Germany [2] Institute of Microbiology, Friedrich Schiller University Jena, Germany [3] Leibniz Research Group – Biobricks of Microbial Natural Product Syntheses, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany [4] Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark * Corresponding author: [email protected]
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Figure Legends Figure S1. Expression analysis of tynC in strains analyzed by reverse transcription polymerase chain reaction (RT-PCR). Expected signal for tynC = 208 bp (for both cDNA and gDNA). Expected signal for act1 = 121 bp (cDNA) / 199 bp (gDNA); -C1 = control of RT without template; -RT = control of cDNA synthesis without RT; gC = gDNA template; -C2 = control of PCR without template; M = Marker bands, Hyperladder 25 bp (Bioline, 5 µL). DNA was visualized by ethidium bromide (1% (w/v); Roth) on a 3 % (w/v) TAE agarose gel. Please note that gC and –C2 are in inverted order on the act1 specific gel. Figure S2. Alignment of predicted coding DNA sequence (CDS) and gDNA sequence of Af293 and CEA10. Prediction of CDS by AUGUSTUS1 with subsequent alignment by AlignX (Invitrogen, Carlsbad, CA, USA) revealed additional intron (light grey) in CEA10. According to the prediction, the single nucleotide insertion at position 3881 (highlighted in black) resulted in a silent mutation. Figure S3. Southern blot analysis of tynC deletion and subsequent complementation with tynCAf293 in akuBKU80. (A) Overview of construct integration and restriction strategy. (B) Separation of BamHI-restricted DNA showed signals of the expected size for akuBKU80 (4.2 kb), ∆tynC (8.3 kb) and tynCAf293 complemented (4.7 kb). (C) Sequencing of tynC in akuBKU80 and akuBKU80 tynCAf293 comp(lemented) strain confirmed the change of a single adenosine (indicated by arrows). hygR = hygromycin resistance gene; PgpdA = gpdA promoter of A. nidulans; ptrA = pyrithiamine resistance cassette of Aspergillus oryzae; Ttef = tef terminator of Ashbya gossypii; TtrpC = trpC terminator of A. nidulans. Figure S4. MS2 fragmentation pattern for m/z 345 [M + H]+ of different samples in comparison to the trypacidin authentic standard. Similar fragmentation patterns for the standard, and extracts of Af293, akuBKU80 tynCAf293 comp(lemented), and akuBKU80 tetON-cas9 tynC+ for m/z 345 [M + H]+ were observed.
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Figure S5. Southern blot analysis of tetON-cas9 integration at native pyrG locus in akuBKU80. (A) Overview of construct integration and restriction strategy (pyrG = native pyrG locus mutated and dysfunctional). (B) Separation of DNA fragments digested with EcoRV showed the signals expected for the wild-type strain CEA17 ΔakuB pyrG+ (2.8 kb) and transformant with homologously integrated tetON-cas9 construct (9.9 kb). (C) All strains displayed an additional band of 3.5 kb, which resulted from the integrated A. fumigatus pyrG gene into the akuB gene locus. hygR = hygromycin resistance gene; PtrpC = trpC promoter of A. nidulans; PtetON = synthetic tetON promoter cassette; Ttef = tef terminator of A. nidulans; TtrpC = trpC terminator of A. nidulans. Figure S6. Plasmid map of pJW split-ptrA tynC and strategy for the generation of new plasmids targeting any gene of interest. (A) Overview of plasmid pJW split-ptrA tynC, with split-marker cassette (blue) and gRNA cassette. gRNA cassette comprises of gpdA promoter from A. nidulans (PgpdA; grey), pre-gRNA and A. nidulans trpC terminator (term; bright blue). PregRNA encompasses 20 bp spacer region (pink) specific for target gene and ~80 bp gRNA scaffold (black), flanked by hammerhead and HDV ribozymes (yellow-checked and yellow) respectively. ptrA of split-marker cassette (PptrA, ptrA*, term; blue) is separated in two fragments with 500 bp overlaps to each other (blue striped) and interrupted by tynC-specific protospacer and PAM site (pink + bright blue). (B) For the integration of spacer and protospacer of new target site, the plasmid is amplified in two fragments by overlapping primers. Tails of primer encompass sequence for new spacer/protospacer (green) as well as sequence overlaps specific for other DNA fragment. (C) Gibson cloning reassembles plasmid with spacer in gRNA targeting another protospacer of a new gene of interest as well as respective site (protospacer) in the split-marker.
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Figure S1
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Figure S2
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Figure S3
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Figure S4
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Figure S5
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Figure S6
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Table S1. Strains used in this study strain
Decription
source
Af293
Clinical isolate
FGSC A1100
CEA10
Clinical isolate
FGSC A1163 see 2
+
akuBKU80
CEA17 ∆akuB pyrG
akuBKU80 ∆tynC
CEA17 ∆akuB pyrG+; ∆tynC::hph
KU80
tynC
KU80
ON
akuB akuB
Af293
comp(lemented)
tet -cas9
+
CEA17 ∆akuB pyrG ; tynC
This study
comp
This study
ON
This study
CEA17 ∆akuB::pyrG, ∆pyrG::tet ::cas9::hph KU80
akuBKU80 tetON-cas9 tynC+
Af293
ON
akuB tet -cas9 with single nucleotide deletion at pos 3881 + ectopic integration of pJW split-ptrA tynC
This study
Table S2. Primers used in this study Primer # oJW0096 oJW0097 oJW0137 oJW0138 oJW0139 oJW0146 oJW0181 oJW0182 oJW0183 oJW0184 oJW0185 oJW0186 oJW0197 oJW0198 oJW0199 oJW0200 oJW0211 oJW0228 oJW0434 oJW0435 oJW0436 oJW0437
sequence (5'->3') AGCCGAGGGATTCAAGTG GTGGTTCCTGATTGGGATTG ATGCCTGCAGGTCGACTCTAGAGGATCCCCGAGCTAC TCAGACATAGCTA ATACACCGGGCAAAGCGTGGGAATGGAGGGTTATA CCCTCCATTCCCACGCTTTGCCCGGTGTATGAAAC ACCATGATTACGAATTCGAGCTCGGTACCCGGGACTT GGGGCCTATTCAT AGGGCAAAGGAATAGTTTCCTCTTGGAGCAAAAGTG CTTTCTCTTCTTTTTTGGAGGCTCGGGAATTAATTCCGC TTTATCCATTGTGATGTGATGGAGTTGAG ATTCCCGAGCCTCCAAAAAAGAAGAGAAAGGTCGAATT GGGTACCGCCGACAAGAAGTATAGCATCGG TGCTCCAAGAGGAAACTATTCCTTTGCCCTCGGAC GGCGGGACGAGCTTACTCGTTTCGTCCTCACGGACTCAT CAGCCGCCGCGGTGATGTCTGCTCAAGC TGAGGACGAAACGAGTAAGCTCGTCCCGCCGCGGGGAG GAATAACGTTTTAGAGCTAGAAATAGCAAGT AGCTCCAGTTATTCCTCCCCGCGGCGGTGAAGAGCTTGA CATTGGGGA TCTTCACCGCCGCGGGGAGGAATAACTGGAGCTTCTCAGA CCAAGC TTAGGGAGCTTACGCGACGGCGCAAGCTCATG TGAGCTTGCGCCGTCGCGTAAGCTCCCTAATTGGC CAAAATCCCTTAACGTGAGTTACGCGCGGAGCCAAGAGCG GATTCC TCCGCGCGTAACTCACGTTAAGGGATTTTGGTC CACCTCCTGCTGGATCAGA GAGAAACTGGCACTGTCCTTG CCACGTCACCACTTTCAACTC CTGCATACGGTCGGAGATAC
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function tynC seq fw tynC seq rv 5'UTR fw 5'UTR rv tetON fw 3' UTR rv 3' UTR fw tetON SV40 rv SV40-Cas9-hph fw Cas9-hph rv gRNA tynC- akuBKU80 part1 rv gRNA tynC- akuBKU80 part2 fw ampR ptrA-part1 tynC-proto rv tynC-proto ptrA-part2 ori fw proto ptrA-part2 ori rv gRNA cassette fw gRNA cassette rev ampR-ptrA-part1 fw tynC rt-pcr fw tynC rt-pcr rv act1 rt-pcr fw act1 rt-pcr rv
Table S3: Plasmids generated in this study. Plasmid pJW tetON::cas9::hph
pJW split-ptrA tynC
Fragments #1 backbone with E. coli settings #2 5’UTR of A. fumigatus pyrG #3 tetON promoter #4 cas9::Ttef + hph cassette #5 3’UTR of A. fumigatus pyrG
Fragment size 2.7 kb 1.0 kb 2.0 kb 6.0 kb 1.0 kb
Template of fragment pUC18 (SmaI) CEA17 ∆akuB pyrG+ pSK562 3 pFC332 CEA17 ∆akuB pyrG+
#1 ampR + ptrA part 1 #2 ptrA part 2 + ori #3 gRNA tynC part 1 #4 gRNA tynC part 2
3.2 kb 1.9 kb 0.6 kb 0.5 kb
pSK275 4 pSK275 3 pFC334 3 pFC334
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Primer no. oJW0137/138 oJW0139/182 oJW0183/184 oJW0181/146 oJW0228/197 oJW0198/199 oJW0200/185 oJW0186/211
References (1) Stanke, M., Diekhans, M., Baertsch, R., and Haussler, D. (2008) Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 24, 637–44. (2) Da Silva Ferreira, M. E., Kress, M. R., Savoldi, M., Goldman, M. H. S., Härtl, A., Heinekamp, T., Brakhage, A. A., and Goldman, G. H. (2006) The akuBKU80 mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryotic Cell 5, 207–211. (3) Nødvig, C. S., Nielsen, J. B., Kogle, M. E., and Mortensen, U. H. (2015) A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS one 10, e0133085. (4) Szewczyk, E., and Krappmann, S. (2010) Conserved regulators of mating are essential for Aspergillus fumigatus cleistothecium formation. Eukaryotic Cell 9, 774–783.
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Figure Legends Figure S1. Expression analysis of tynC in strains analyzed by reverse transcription polymerase chain reaction (RT-PCR). Expected signal for tynC = 208 bp (for both cDNA and gDNA). Expected signal for act1 = 121 bp (cDNA) / 199 bp (gDNA); -C1 = control of RT without template; -RT = control of cDNA synthesis without RT; gC = gDNA template; -C2 = control of PCR without template; M = Marker bands, Hyperladder 25 bp (Bioline, 5 µL). DNA was visualized by ethidium bromide (1% (w/v); Roth) on a 3 % (w/v) TAE agarose gel. Please note that gC and –C2 are in inverted order on the act1 specific gel. Figure S2. Alignment of predicted coding DNA sequence (CDS) and gDNA sequence of Af293 and CEA10. Prediction of CDS by AUGUSTUS1 with subsequent alignment by AlignX (Invitrogen, Carlsbad, CA, USA) revealed additional intron (light grey) in CEA10. According to the prediction, the single nucleotide insertion at position 3881 (highlighted in black) resulted in a silent mutation. Figure S3. Southern blot analysis of tynC deletion and subsequent complementation with tynCAf293 in akuBKU80. (A) Overview of construct integration and restriction strategy. (B) Separation of BamHI-restricted DNA showed signals of the expected size for akuBKU80 (4.2 kb), ∆tynC (8.3 kb) and tynCAf293 complemented (4.7 kb). (C) Sequencing of tynC in akuBKU80 and akuBKU80 tynCAf293 comp(lemented) strain confirmed the change of a single adenosine (indicated by arrows). hygR = hygromycin resistance gene; PgpdA = gpdA promoter of A. nidulans; ptrA = pyrithiamine resistance cassette of Aspergillus oryzae; Ttef = tef terminator of Ashbya gossypii; TtrpC = trpC terminator of A. nidulans. Figure S4. MS2 fragmentation pattern for m/z 345 [M + H]+ of different samples in comparison to the trypacidin authentic standard. Similar fragmentation patterns for the standard, and extracts of Af293, akuBKU80 tynCAf293 comp(lemented), and akuBKU80 tetON-cas9 tynC+ for m/z 345 [M + H]+ were observed.
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Figure S5. Southern blot analysis of tetON-cas9 integration at native pyrG locus in akuBKU80. (A) Overview of construct integration and restriction strategy (pyrG = native pyrG locus mutated and dysfunctional). (B) Separation of DNA fragments digested with EcoRV showed the signals expected for the wild-type strain CEA17 ΔakuB pyrG+ (2.8 kb) and transformant with homologously integrated tetON-cas9 construct (9.9 kb). (C) All strains displayed an additional band of 3.5 kb, which resulted from the integrated A. fumigatus pyrG gene into the akuB gene locus. hygR = hygromycin resistance gene; PtrpC = trpC promoter of A. nidulans; PtetON = synthetic tetON promoter cassette; Ttef = tef terminator of A. nidulans; TtrpC = trpC terminator of A. nidulans. Figure S6. Plasmid map of pJW split-ptrA tynC and strategy for the generation of new plasmids targeting any gene of interest. (A) Overview of plasmid pJW split-ptrA tynC, with split-marker cassette (blue) and gRNA cassette. gRNA cassette comprises of gpdA promoter from A. nidulans (PgpdA; grey), pre-gRNA and A. nidulans trpC terminator (term; bright blue). PregRNA encompasses 20 bp spacer region (pink) specific for target gene and ~80 bp gRNA scaffold (black), flanked by hammerhead and HDV ribozymes (yellow-checked and yellow) respectively. ptrA of split-marker cassette (PptrA, ptrA*, term; blue) is separated in two fragments with 500 bp overlaps to each other (blue striped) and interrupted by tynC-specific protospacer and PAM site (pink + bright blue). (B) For the integration of spacer and protospacer of new target site, the plasmid is amplified in two fragments by overlapping primers. Tails of primer encompass sequence for new spacer/protospacer (green) as well as sequence overlaps specific for other DNA fragment. (C) Gibson cloning reassembles plasmid with spacer in gRNA targeting another protospacer of a new gene of interest as well as respective site (protospacer) in the split-marker.
3
Figure S1
4
Figure S2
5
Figure S3
6
Figure S4
7
Figure S5
8
Figure S6
9
Table S1. Strains used in this study strain
Decription
source
Af293
Clinical isolate
FGSC A1100
CEA10
Clinical isolate
FGSC A1163 see 2
+
akuBKU80
CEA17 ∆akuB pyrG
akuBKU80 ∆tynC
CEA17 ∆akuB pyrG+; ∆tynC::hph
KU80
tynC
KU80
ON
akuB akuB
Af293
comp(lemented)
tet -cas9
+
CEA17 ∆akuB pyrG ; tynC
This study
comp
This study
ON
This study
CEA17 ∆akuB::pyrG, ∆pyrG::tet ::cas9::hph KU80
akuBKU80 tetON-cas9 tynC+
Af293
ON
akuB tet -cas9 with single nucleotide deletion at pos 3881 + ectopic integration of pJW split-ptrA tynC
This study
Table S2. Primers used in this study Primer # oJW0096 oJW0097 oJW0137 oJW0138 oJW0139 oJW0146 oJW0181 oJW0182 oJW0183 oJW0184 oJW0185 oJW0186 oJW0197 oJW0198 oJW0199 oJW0200 oJW0211 oJW0228 oJW0434 oJW0435 oJW0436 oJW0437
sequence (5'->3') AGCCGAGGGATTCAAGTG GTGGTTCCTGATTGGGATTG ATGCCTGCAGGTCGACTCTAGAGGATCCCCGAGCTAC TCAGACATAGCTA ATACACCGGGCAAAGCGTGGGAATGGAGGGTTATA CCCTCCATTCCCACGCTTTGCCCGGTGTATGAAAC ACCATGATTACGAATTCGAGCTCGGTACCCGGGACTT GGGGCCTATTCAT AGGGCAAAGGAATAGTTTCCTCTTGGAGCAAAAGTG CTTTCTCTTCTTTTTTGGAGGCTCGGGAATTAATTCCGC TTTATCCATTGTGATGTGATGGAGTTGAG ATTCCCGAGCCTCCAAAAAAGAAGAGAAAGGTCGAATT GGGTACCGCCGACAAGAAGTATAGCATCGG TGCTCCAAGAGGAAACTATTCCTTTGCCCTCGGAC GGCGGGACGAGCTTACTCGTTTCGTCCTCACGGACTCAT CAGCCGCCGCGGTGATGTCTGCTCAAGC TGAGGACGAAACGAGTAAGCTCGTCCCGCCGCGGGGAG GAATAACGTTTTAGAGCTAGAAATAGCAAGT AGCTCCAGTTATTCCTCCCCGCGGCGGTGAAGAGCTTGA CATTGGGGA TCTTCACCGCCGCGGGGAGGAATAACTGGAGCTTCTCAGA CCAAGC TTAGGGAGCTTACGCGACGGCGCAAGCTCATG TGAGCTTGCGCCGTCGCGTAAGCTCCCTAATTGGC CAAAATCCCTTAACGTGAGTTACGCGCGGAGCCAAGAGCG GATTCC TCCGCGCGTAACTCACGTTAAGGGATTTTGGTC CACCTCCTGCTGGATCAGA GAGAAACTGGCACTGTCCTTG CCACGTCACCACTTTCAACTC CTGCATACGGTCGGAGATAC
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function tynC seq fw tynC seq rv 5'UTR fw 5'UTR rv tetON fw 3' UTR rv 3' UTR fw tetON SV40 rv SV40-Cas9-hph fw Cas9-hph rv gRNA tynC- akuBKU80 part1 rv gRNA tynC- akuBKU80 part2 fw ampR ptrA-part1 tynC-proto rv tynC-proto ptrA-part2 ori fw proto ptrA-part2 ori rv gRNA cassette fw gRNA cassette rev ampR-ptrA-part1 fw tynC rt-pcr fw tynC rt-pcr rv act1 rt-pcr fw act1 rt-pcr rv
Table S3: Plasmids generated in this study. Plasmid pJW tetON::cas9::hph
pJW split-ptrA tynC
Fragments #1 backbone with E. coli settings #2 5’UTR of A. fumigatus pyrG #3 tetON promoter #4 cas9::Ttef + hph cassette #5 3’UTR of A. fumigatus pyrG
Fragment size 2.7 kb 1.0 kb 2.0 kb 6.0 kb 1.0 kb
Template of fragment pUC18 (SmaI) CEA17 ∆akuB pyrG+ pSK562 3 pFC332 CEA17 ∆akuB pyrG+
#1 ampR + ptrA part 1 #2 ptrA part 2 + ori #3 gRNA tynC part 1 #4 gRNA tynC part 2
3.2 kb 1.9 kb 0.6 kb 0.5 kb
pSK275 4 pSK275 3 pFC334 3 pFC334
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Primer no. oJW0137/138 oJW0139/182 oJW0183/184 oJW0181/146 oJW0228/197 oJW0198/199 oJW0200/185 oJW0186/211
References (1) Stanke, M., Diekhans, M., Baertsch, R., and Haussler, D. (2008) Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 24, 637–44. (2) Da Silva Ferreira, M. E., Kress, M. R., Savoldi, M., Goldman, M. H. S., Härtl, A., Heinekamp, T., Brakhage, A. A., and Goldman, G. H. (2006) The akuBKU80 mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryotic Cell 5, 207–211. (3) Nødvig, C. S., Nielsen, J. B., Kogle, M. E., and Mortensen, U. H. (2015) A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS one 10, e0133085. (4) Szewczyk, E., and Krappmann, S. (2010) Conserved regulators of mating are essential for Aspergillus fumigatus cleistothecium formation. Eukaryotic Cell 9, 774–783.
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