Supporting information
Supplementary Methods
DNA manipulation
Escherichia coli DH5α was used for DNA manipulation. The polymerase chain reaction (PCR) was
performed using PrimesSTAR HS DNA polymerase (TaKaRa, Otsu, Japan). The In-Fusion HD
Cloning Kit (Clontech, Palo Alto, CA) was used for plasmid construction.
Media
Transformants were selected on Czapek-Dox (CD) medium (0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4,
0.05% MgSO4•7H2O, 0.002% FeSO4•7H2O, and 2% dextrin, pH 5.5). CD medium supplemented with
0.5% uridine and 0.2% uracil was used for the growth of candidate transformants for pyrG mutants.
Potato dextrose (PD) medium (Nissui, Tokyo, Japan) was used for the observation of conidial color.
DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH 2PO4, 0.05% MgSO4•7H2O,
pH 5.5) was used for the preparation of cell extracts.
Preparation of protein extracts and western blot analysis
A. oryzae mycelia grown in DPY liquid medium were frozen by liquid nitrogen and homogenized using
multi-beads shocker (Yasui Kikai, Osaka, Japan). The homogenized mycelia were suspended in
extraction buffer (50 mM Tris-HCl [pH 7.5], 1 mM phenylmethylsulfonyl fluoride, and 1% protease
inhibitor cocktail (Sigma, St. Louis, MO)), and debris was removed by centrifugation at 1,000 × g for 10
min. Protein extracts were separated by electrophoresis on an 8% polyacrylamide gel containing sodium
dodecyl sulfate, and then blotted onto Immobilon Transfer Membranes (Millipore, Bedford, MA). For
Cas9 detection, the membrane was incubated with monoclonal ANTI-FLAG® M2 antibody (Sigma;
1:5,000 dilution) as a primary antibody and with 0.2 μg/mL peroxidase-labeled anti-mouse IgG (H+L)
(Vector Laboratories, Burlingame, CA) as a secondary antibody. FLAG-tagged Cas9 was detected with a
Western Lightning-ECL system (PerkinElmer, Waltham, MA) and the luminescent image analyzer
LAS-4000miniEPUV (Fuji Photo Film, Tokyo, Japan).
Supplementary Material. Sequences of sgRNA, U6 promoter, U6 terminator, and
codon-optimized cas9
Codon-optimized cas9
(Small letters and underlines indicate the FLAG-tag and SV40NLS encoding sequences, respectively.)
ATGgattacaaggatgacgacgataagATCATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAG
TCCCAGCAGCCGACAAGAAGTACTCCATCGGTCTCGACATCGGTACCAACTCCGTCGGTTGG
GCCGTCATCACCGACGAGTACAAGGTCCCCTCCAAGAAGTTCAAGGTCCTCGGTAACACCG
ACCGTCACTCCATCAAGAAGAACCTCATCGGTGCCCTCCTCTTCGACTCCGGTGAGACCGCC
GAGGCCACCCGTCTCAAGCGTACCGCCCGTCGTCGTTACACCCGTCGTAAGAACCGTATCTG
CTACCTCCAGGAGATCTTCTCCAACGAGATGGCTAAGGTCGATGACTCCTTCTTCCACCGTCT
CGAGGAGTCCTTCCTCGTCGAGGAGGATAAGAAGCACGAGCGTCACCCCATCTTCGGTAAC
ATCGTCGACGAGGTCGCCTACCACGAGAAGTACCCCACCATCTACCACCTCCGTAAGAAGCT
TGTCGACTCTACTGATAAGGCTGACCTTCGTCTCATCTACCTCGCCCTCGCCCACATGATCAA
GTTCCGTGGTCACTTCCTCATCGAGGGTGACCTCAACCCTGACAACTCCGATGTCGACAAGC
TCTTCATCCAGCTCGTTCAGACTTACAACCAGCTCTTCGAAGAGAACCCCATCAACGCTTCC
GGTGTTGACGCCAAGGCCATCCTCTCCGCTCGTCTCTCCAAGTCCCGTCGTCTCGAGAACCT
CATCGCTCAGCTCCCTGGTGAGAAGAAGAACGGTCTCTTCGGTAACCTTATCGCCCTCTCTC
TCGGTCTCACCCCCAACTTCAAGTCCAACTTCGACCTCGCCGAGGATGCCAAGCTTCAGCTC
TCCAAGGACACCTACGATGACGATCTCGACAACCTCCTCGCCCAGATCGGTGACCAGTACGC
TGACCTCTTCCTTGCCGCTAAGAACCTCTCCGACGCCATCCTCCTCTCCGATATCCTCCGTGT
CAACACCGAGATCACCAAGGCTCCCCTCTCCGCTTCTATGATCAAGCGTTACGATGAGCACC
ACCAGGACCTCACTCTTCTCAAGGCCCTTGTCCGTCAGCAGCTCCCTGAGAAGTACAAGGA
GATCTTCTTCGATCAGTCCAAGAACGGTTACGCCGGTTACATCGACGGCGGTGCTTCCCAGG
AGGAGTTCTACAAGTTCATCAAGCCCATCCTCGAGAAGATGGACGGTACCGAGGAGCTCCT
CGTCAAGCTCAACCGTGAGGATCTCCTCCGTAAGCAGCGTACTTTCGACAACGGTTCCATCC
CCCACCAGATCCACCTTGGCGAACTCCACGCTATCCTCCGTCGTCAGGAGGACTTCTACCCC
TTCCTCAAGGACAACCGTGAGAAGATCGAGAAGATCCTCACCTTCCGTATCCCCTACTACGT
TGGCCCCCTCGCCCGTGGTAACTCCCGTTTCGCCTGGATGACTCGTAAGTCCGAAGAGACCA
TCACTCCCTGGAACTTCGAGGAAGTCGTCGATAAGGGTGCCTCTGCCCAGTCCTTCATCGAG
CGTATGACTAACTTCGACAAGAACCTCCCTAACGAGAAGGTCCTTCCTAAGCACTCTCTCCT
TTACGAGTACTTCACTGTTTACAACGAGCTCACCAAGGTCAAGTACGTCACCGAAGGTATGC
GTAAGCCCGCCTTCCTCTCTGGTGAGCAGAAGAAGGCTATCGTCGACCTCCTCTTCAAGACC
AACCGTAAGGTTACCGTCAAGCAGCTCAAGGAAGACTACTTCAAGAAGATCGAGTGCTTCG
ACTCCGTTGAGATCTCCGGTGTCGAGGATCGTTTCAACGCTTCCCTCGGTACCTACCACGATC
TCCTCAAGATCATCAAGGACAAGGACTTCCTCGACAACGAGGAGAACGAGGACATCCTTGA
GGACATCGTCCTCACCCTTACCCTCTTCGAGGATCGTGAGATGATCGAAGAACGTCTCAAGA
CTTACGCTCACCTCTTCGACGACAAGGTCATGAAGCAGCTCAAGCGTCGTCGTTACACTGGT
TGGGGCCGTCTCTCCCGTAAGCTGATCAACGGCATCCGTGACAAGCAGTCTGGTAAGACTAT
CCTCGACTTCCTTAAGTCCGATGGTTTCGCCAACCGTAACTTCATGCAGCTCATCCACGACG
ACTCTCTCACCTTCAAGGAGGACATCCAGAAGGCTCAGGTTTCCGGTCAGGGTGACTCCCTT
CACGAGCACATCGCTAACCTCGCCGGTTCCCCCGCTATCAAGAAGGGTATCCTCCAGACCGT
TAAGGTCGTCGATGAGCTCGTCAAGGTTATGGGTCGTCACAAGCCCGAGAACATCGTTATCG
AGATGGCCCGTGAGAACCAGACTACCCAGAAGGGTCAGAAGAACTCTCGTGAACGTATGAA
GCGTATCGAGGAGGGTATCAAGGAACTCGGCTCCCAGATCCTTAAGGAGCACCCCGTCGAG
AACACCCAGCTTCAGAACGAGAAGCTCTACCTCTACTACCTCCAGAACGGCCGTGACATGTA
CGTTGATCAGGAGCTCGACATCAACCGTCTCTCCGACTACGACGTCGATCACATCGTCCCCC
AGTCCTTCCTCAAGGATGACTCTATCGACAACAAGGTCCTCACCCGTTCCGATAAGAACCGT
GGCAAGTCTGACAACGTCCCCTCCGAAGAGGTTGTCAAGAAGATGAAGAACTACTGGCGTC
AGCTTCTCAACGCCAAGCTCATCACCCAGCGTAAGTTCGATAACCTCACTAAGGCTGAACGT
GGTGGCCTCTCTGAGCTCGACAAGGCCGGCTTCATCAAGCGTCAGCTTGTTGAGACTCGTCA
GATCACCAAGCACGTCGCCCAGATTCTCGACTCTCGCATGAACACCAAGTACGATGAGAAC
GACAAGCTCATCCGTGAGGTCAAGGTTATCACTCTCAAGTCTAAGCTCGTCTCCGACTTCCG
TAAGGACTTCCAGTTCTACAAGGTCCGTGAGATCAACAACTACCACCACGCCCACGATGCCT
ACCTCAACGCTGTCGTTGGCACTGCTCTTATCAAGAAGTACCCCAAGCTTGAGTCCGAGTTC
GTCTACGGTGACTACAAGGTCTACGATGTTCGTAAGATGATCGCTAAGTCCGAGCAGGAGAT
CGGTAAGGCCACCGCTAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGA
TCACCCTCGCCAACGGTGAGATCCGTAAGCGTCCCCTCATCGAGACCAACGGTGAGACTGG
TGAGATCGTCTGGGACAAGGGTCGTGACTTCGCCACTGTCCGTAAGGTCCTCTCCATGCCCC
AGGTCAACATCGTCAAGAAGACCGAGGTCCAGACCGGTGGCTTCTCCAAGGAATCTATCCT
CCCCAAGCGTAACTCCGACAAGCTCATCGCTCGCAAGAAGGACTGGGACCCCAAGAAGTAC
GGCGGTTTCGACTCTCCCACTGTCGCTTACTCCGTCCTCGTTGTCGCCAAGGTCGAGAAGGG
TAAGTCTAAGAAGCTCAAGTCCGTCAAGGAGCTCCTCGGCATCACCATCATGGAGCGTTCCT
CCTTCGAGAAGAACCCCATCGACTTCCTCGAGGCCAAGGGTTACAAGGAGGTCAAGAAGGA
CCTCATCATCAAGCTTCCCAAGTACTCCCTCTTCGAGCTTGAGAACGGTCGTAAGCGTATGCT
CGCTTCCGCCGGTGAGCTCCAGAAGGGTAACGAGCTCGCTCTCCCCTCCAAGTACGTCAAC
TTCCTCTACCTCGCCTCCCACTACGAGAAGCTCAAGGGTTCTCCCGAAGACAACGAGCAGA
AGCAGCTCTTCGTCGAGCAGCACAAGCACTACCTTGATGAGATCATCGAGCAGATCTCCGAG
TTCTCCAAGCGTGTCATCCTCGCCGACGCTAACCTCGATAAGGTCCTCTCCGCTTACAACAA
GCACCGTGACAAGCCCATCCGTGAGCAGGCTGAGAACATCATCCACCTCTTCACTCTCACCA
ACCTTGGTGCCCCTGCTGCCTTCAAGTACTTCGACACCACCATCGACCGTAAGCGTTACACC
TCTACCAAGGAGGTCCTCGACGCCACTCTCATCCACCAGTCCATTACCGGTCTCTACGAGAC
TCGTATCGACCTCTCTCAGCTCGGTGGTGACTCCCGTGCTGACCCCAAGAAGAAGCGTAAG
GTCTGA
U6 promoter
TGGTTCACTTCTCTTTAGAAATCAACTGTGGGTTTTGCTTTTTGCTTCATTCTCTTTGTCTTCT
CCATCTTTGATCAAATCCTGGACTTTCTCAATCCCCAGCTAATTCAATCATAGTCAGTTTTCTA
TTTTTATTATTTCTTTTTCTTTTGAAATGTGATTAACAACCAGTCCGTTATATATCTTGTACCCA
GATTACGCCCAACTCGTGCTCCTCAGCCACAAAGATACTCAATTGATAGCCAAGATACATACA
TACCACAAAGTAAGGACTCCATGCATTGAGTATTACTCATCGTATTCTAGACTACTCCAAAAC
TCAGCACATAGACAAACAATACGAACCTCGTCTAGGGGTGATTCAGAGGCGGCAAAGCGGG
GTTTTCGCATTTGATGTTCCTGGCACTTATGTAAGCCCACGCTTCCCGCTCAACTAAACCATC
AGCCAATCAGACTGCTCAGATTTATCTTTTGAAGGGTAAATAAATCATTGTAAAGAAGAACA
AGT
U6 terminator
TTTTTTTTTGAGCATTTATCAGCTTGATATAGAGGTAGGAATGTATGGAGGTGCAGAATGGCTA
TTTTGTTATTGGAGCGGGTTCGAAACGGAGGGCAGGAGACTTTTTCTAAATACGTCACGTGA
TATAGAGCTGCT
sgRNA
(protospacer sequence)-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU
AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC
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Supplementary Table 1. Primers used in this study
Primer name
Primer name Sequence 5' to 3'
Primers to amplify codon-optimized cas9
CAS9-in-fusion-6R
CTACTACAGATCCCCTCAGACCTTACGCTTCTTCTTGGGGTC
FLAG-NLS-cas9-F
TGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACTCCATCGGTC
IF5-FLAG-NLS-F
TCGAGCTCGGTACCCATGGATTACAAGGATGACGACGATAAGATCATGGCCCCAAAGAAGAAGCGG
Primers to amplify the U6 promoter and terminator and sgRNA sequence
pUNA+U6p-F
ATAGGAAAGGATCCTTAATGCCGGCTCATTCAAACGGAAATACGAGGGAC
gRNA+U6t-F1
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG
gRNA+U6t-R1
ACATTCCTACCTCTATATCAAGCTGATAAATGCTCAAAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGA
gRNA+U6t-F2
TCAGCTTGATATAGAGGTAGGAATGTATGGAGGTGCAGAATGGCTATTTTGTTATTGGAGCGGGTTCGAAACGGA
gRNA+U6t-R2
AGCAGCTCTATATCACGTGACGTATTTAGAAAAAGTCTCCTGCCCTCCGTTTCGAACCCGCTCCAATAACAAAAT
pUNA+U6t-R3
GCAGGTCGACTCTAGAGCAGCTCTATATCACGTGACGTATTTAGA
Primers with protospacer sequences to amplify the U6 promoter
gwA1-PU6-R
GTGACGCGCCAGTAGATCCACACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG
gwA2-PU6-R
GCTTATGGCTCTTAGACAGCCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG
gwA3-PU6-R
ATAAGCTGCGAGGCATCTTTCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG
gwA4-PU6-R
GTGCGCGGGTCTTCTTAGTTCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG
gyA-PU6-R
TTAGTGAGAATCATTTGGCGCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG
gpG-PU6-R
CTCGGAGCCGTAGGGGAAGTCCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG
Primers with protospacer sequences to amplify the U6 terminator
gwA1-TU6-F
GTGGATCTACTGGCGCGTCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
gwA2-TU6-F
GGCTGTCTAAGAGCCATAAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
gwA3-TU6-F
GAAAGATGCCTCGCAGCTTATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
gwA4-TU6-F
GAACTAAGAAGACCCGCGCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
gyA-TU6-F
GCGCCAAATGATTCTCACTAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
gpG-TU6-F
GACTTCCCCTACGGCTCCGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC