Supplementary Information

Supplementary Information Supplementary Table 1. Strain table Strain name Genotype Reference CEN.PK111-27B MATa leu2 trp1 Euroscarf TC-49 TC-50 pdc5::aro4* aro10::aro7* This study TC-50 MATa leu2 trp1 + P414 - TRP1-TEF1p-Cas9-CYC1t This study TC-71 TC-50 ade2::crtyb This study TC-72 TC-50 his3::crte This study TC-73 TC-50 ura3::crti This study TC-74 TC-50 ade2::crtyb ura3::crti his3::crte This study Supplementary Table 2. Plasmid table Plasmid name Description Reference p414 TRP1-TEF1p-Cas9-CYC1t Addgene ptajak-96 pesc-leu TJOS-97F-TJOS-97R This study ptajak-97 ptajak-96 grna.ade2 This study ptajak-98 ptajak-96 grna.ura3 This study ptajak-99 ptajak-96 grna.his3 This study ptajak-101 ptajak-96 grna.pdc5-grna.aro10 This study ptajak-103 ptajak-96 grna.ade2-grna.ura3-grna.his3 This study ptajak-104 ptajak-96 grna.his3-2 This study ptajak-105 ptajak-96 grna.ade2-grna.ura3-grna.his3-2 This study Supplementary Table 3. List of primers Primer name ASR_X001 ASR_X002 ASR_X003 ASR_X004 ASR_X005 ASR_X006 ASR_X007 ASR_X008 Sequence TCTTAACCCAACTGCACAGAA GACCCCATTCTTTGAAGGTACTTCTG ACAATTCTGCTAACATCAAAAG CCTTTTGATGTTAGCAGAATTGTCA GGAAGTACCTTCAAAGAATGG CTGTGAGGATGTTCGCGTAATCCAT TGTTTTATATTTGTTGTAAAAAGTAG ATAA TACTTTTTACAACAAATATAAAACA ATGGATTACGCGAACATCCTC TCCTTCCTTTTCGGTTAGAGCGGATT CACAGAGGGATATCGGCTAG AAAGCTAGCCGATATCCCTCTGTGA ATCCGCTCTAACCGAAAAGGA GTCAACAGTACCCTTAGTATATTCTT TCTCAAGCAAGGTTTTCAGTATAAT Description Forward primer for URA3 upstream Reverse primer for URA3 upstream with PGK1p Forward primer for PGK1p with URA3 Reverse primer for PGK1p with crte Forward primer for crte with PGK1p Reverse primer for crte with tcyc1 Forward primer for tcyc1 with crte Reverse primer for tcyc1 with URA3

ASR_X009 ASR_X010 ASR_X015 ASR_X016 ASR_X017 ASR_X018 ASR_X019 ASR_X020 ASR_X021 ASR_X022 ASR_X023 ASR_X024 ASR_X123 ASR_X124 ASR_X125 ASR_X032 ASR_X033 ASR_X034 ASR_X035 ASR_X126 ASR_X127 G TTATACTGAAAACCTTGCTTGAGAA AGAATATACTAAGGGTACTGTTGAC ATTG CTGGCGAGGTATTGGATAGTT TCTTGGCCTCCTCTAGTACACTCT TTTAAAAGCTTGACCGAGAGC GATTGCTCTCGGTCAAGCTTTTAAA TATCCTGATCTTGTTCTTTTCCCATT TTGTTTGTTTATGTGTGTTTATTCGA GAATAAACACACATAAACAAACAA AATGGGAAAAGAACAAGATCAGG AAATCATAAATCATAAGAAATTCGC TCAGAAAGCAAGAACACCAACG ATCCGTTGGTGTTCTTGCTTTCTGAG CGAATTTCTTATGATTTATGATTTTT ATT AAATCCTGATCCAAACCTTTTTACTC ATGCCGGTAGAGGTGTG TATTGACCACACCTCTACCGGCATG AGTAAAAAGGTTTGGATCAGGATTT AAGCAAAAAGAAAAAAGGAAAGC CTGTAATGTAGTTCAGCCTTTGTTC AACATTTTGAAGCTATGGTGTGTGC CAAGGACAATATTTGTGACTTATGT T ACATAAGTCACAAATATTGTCCTTG GCACACACCATAGCTTCAAA TCTGGTAATATGCGAGAGCCGTCAT TTGTAATTAAAACTTAGATTAGATT GCTATG AATCTAATCTAAGTTTTAATTACAA ATGACGGCTCTCGCATATTA TCCTTCCTTTTCGGTTAGAGCGGATT TACTGCCCTTCCCATCCGC CATGAGCGGATGGGAAGGGCAGTA AATCCGCTCTAACCGAAAAGGA TTCTTACCCAATTGTAGAGACTATCT TCTCAAGCAAGGTTTTCAGTATAAT G TTATACTGAAAACCTTGCTTGAGAA GATAGTCTCTACAATTGGGTAAGAA Forward primer for URA3 downstream with tcyc1 Reverse primer for URA3 downstream Forward primer for HIS3 upstream Reverse primer for HIS3 upstream with TDH3p HIS3 Reverse primer for TDH3p with crti Forward primer for crti with TDH3p Reverse primer for crti with tadh1 Forward primer for tadh1 with crti Reverse primer for tadh1 with HIS3 Forward primer for HIS3 downstream with tadh1 Reverse primer for HIS3 downstream Forward primer for ADE2 upstream Reverse primer for ADE2 upstream with TEF1p Forward primer for TEF1p with ADE2 Reverse primer for TEF1p with crtyb Forward primer for crtyb with TEF1p Reverse primer for crtyb with tcyc1 Forward primer for tcyc1 with crtyb Reverse primer for tcyc1 with ADE2 Forward primer for ADE2 downstream,

AAC with tcyc1 ASR_X128 ACCACGTTAATGGCTCCTT Reverse primer for ADE2 downstream ASR_X043 TACCTAAACATCTATAACCTTCAAA AGTA Forward primer for PDC5 upstream ASR_X044 AACATTTTGAAGCTATGGTGTGTGC ATTGGGTGCTACTATGGC Reverse primer for PDC5 upstream with TEF1p ASR_X045 TTCAGCGGCCATAGTAGCACCCAAT GCACACACCATAGCTTCAAA Forward primer for TEF1p with PDC5 ASR_X046 CAGCGAACATTGGAGATTCACTCAT TTGTAATTAAAACTTAGATTAGATT GCTATG Reverse primer for TEF1p with Aro4pm ASR_X047 AATCTAATCTAAGTTTTAATTACAA ATGAGTGAATCTCCAATGTTCG Forward primer for Aro4pm with TEF1p ASR_X048 AAATCATAAATCATAAGAAATTCGC CTATTTCTTGTTAACTTCTCTTCTTTG TCTG Reverse primer for Aro4pm with tadh1 ASR_X049 AAGAAGAGAAGTTAACAAGAAATA GGCGAATTTCTTATGATTTATGATTT TTATT Forward primer for tadh1 with Aro4pm ASR_X050 TTCCATTGGTTTCACAGTCGGCGCTG AGCGACCTCATGCTATACCTG Reverse primer for tadh1 with PDC5 Forward primer for PDC5 TCTCAGGTATAGCATGAGGTCGCTC downstream with AGCGCCGACTGTGAAACC ASR_X051 tadh1 Reverse primer for PDC5 AACACCCAAGATACGGTGG ASR_X052 downstream Forward primer for ARO10 TACCTATCAGTCAGTACGTCTCCAA ASR_X053 upstream Reverse primer for ARO10 upstream with TDH3p GTTGGTGAATTAAGCGCCTT ASR_X054 ASR_X055 CGTTCAAGGCGCTTAATTCACCAAC ARO10 ASR_X056 CAGTTTCTGGTTTTGTGAAATCCATT TTGTTTGTTTATGTGTGTTTATTCGA Reverse primer for TDH3p with Aro7* ASR_X057 GAATAAACACACATAAACAAACAA AATGGATTTCACAAAACCAGAAAC Forward primer for Aro7* with TDH3p ASR_X063 AGCGGGTATATACTTTCATATGTGA TTACTCTTCCAACCTTCTTAGCAAG Reverse primer for Aro7* with tvps13 ASR_X064 CTTGCTAAGAAGGTTGGAAGAGTAA TCACATATGAAAGTATATACCCGC Forward primer for tvps13 with Aro7* ASR_X065 ATTGGCTGTTTAATAACCACGTATG AAGAAAGTATAGCTCAACTATATTC CTT Reverse primer for tvps13 with ARO10 ASR_X066 GAATATAGTTGAGCTATACTTTCTTC ATACGTGGTTATTAAACAGCCA Forward primer for ARO10 downstream with tvps13 ASR_X062 TTAAGATATAAACTGGGCTAGTTTG Reverse primer for ARO10

ASR_X133 ASR_X134 ASR_X136 ASR_X143 ASR_X145 ASR_X146 ASR_X151 ASR_X152 ASR_X153 ASR_X154 ASR_X155 ASR_X156 ASR_X157 C TGATGCATTACCTTGTCATCTTCA GCGGGATTGCTCTCGGTC AAAGCTTGACCGAGAGCAATCCCGC TAGACGACCATCACACCACTGAAGA CATGCCGGTAGAGGTGTG TATTGACCACACCTCTACCGGCATG TCTTCAGTGGTGTGATGGTC TTTTCTTTTTCTATTACTCTTGGCCT TGACAATTCTGCTAACATCAAAAG CCTTTTGATGTTAGCAGAATTGTCA GTCAACAGTACCCTTAGTATATTCTC ATGCCGGTAGAGGTGTG TATTGACCACACCTCTACCGGCATG AGAATATACTAAGGGTACTGTTGAC ATTG GACCCCATTCTTTGAAGGTACTTCTT TTAAAAGCTTGACCGAGAGC GATTGCTCTCGGTCAAGCTTTTAAA GGAAGTACCTTCAAAGAATGG AAATCCTGATCCAAACCTTTTTACTT TCTCAAGCAAGGTTTTCAGTATAAT G TTATACTGAAAACCTTGCTTGAGAA ASR_X158 AGTAAAAAGGTTTGGATCAGGATTT ASR_X011 GAAGAAACATGAAATTGCCCAG ASR_X014 GTCTTATTGTTCTTGATTTGTGCC ASR_X025 ACTCTTGGCCTCCTCTAG ASR_X028 CCGTTCCTCCATCTCTTTTATAT ASR_X129 CACTTCAAATGGAACGCCAA ASR_X130 AGACGGTAATACTAGATGCTGA ASR_X159 TCTCGAGTTCAAGAGAAAAAAAAAG downstream Forward primer for new HIS3 upstream Reverse primer for new HIS3 upstream with TDH3p new HIS3 Reverse primer for tadh1 with new HIS3 Forward primer for new HIS3 downstream with tadh1 Reverse primer for new HIS3 downstream Reverse primer for URA3 upstream with TDH3p URA3 Reverse primer for tadh1 with URA3 Forward primer for URA3 downstream with tadh1 Reverse primer for HIS3 upstream with PGK1p Forward primer for PGK1p with HIS3 Reverse primer for tcyc1 with HIS3 Reverse primer for HIS3 upstream with tcyc1 Forward primer for URA3 Reverse primer for URA3 Forward primer for HIS3 Reverse primer for HIS3 Forward primer for ADE2_new Reverse primer for ADE2_new Forward primer for new HIS3

ASR_X160 TGCCAGGTATCGTTTGAACA Reverse primer for new HIS3 ASR_X067 GACAGGACAGAAAAGTAATTACAAGA Forward primer for PDC5 ASR_X068 ATGACAAGAACTCTGTCGTC Reverse primer for PDC5/ARO4* ASR_X069 GCTGCTGTCAGACAAAGAA Forward primer for ARO4*/PDC5 ASR_X070 GATGTCAAGGCTGAAACTAAGAAG Reverse primer for PDC5 ASR_X071 CATATAAGTTGGGTTTGACTCATCAA Forward primer for ARO10 ASR_X072 GGATGGTTTGCCTCATAAACT Reverse primer for ARO10/ARO7* ASR_X073 TTATACCTATCACTAAGGAAGTTGA Forward primer for ARO7*/ARO10 downstream colony PCR ASR_X074 TCGGTATGTAATAGGTTAGTGG Reverse primer for ARO10 ASR_X012 GAGTAAACTCGAGTGGAATTGC Reverse primer for crte upstream colony PCR ASR_X013 TGGAAGCGATCCTGAAAAAG Forward primer for crte ASR_X026 CCACGATGATAGCTGTGG Reverse primer for crti upstream colony PCR ASR_X027 GGTATACCTTTTGGTGTTGTTG Forward primer for crti ASR_X040 AGCGGTAAATAGGTAGATAATGG Reverse primer for crtyb ASR_X041 GAAAGTCTTGAGTGTGGTCA Forward primer for crtyb TJOS-62 CGTGCGAUAGGGAACAAAAGCTGG Described in Methods AGCT TJOS-63 AGTGCAGGUAGGGAACAAAAGCTG Described in Methods GAGCT TJOS-64 ATCTGTCAUAGGGAACAAAAGCTGG Described in Methods AGCT TJOS-65 CACGCGAUTAACTAATTACATGACT Described in Methods CGA TJOS-66 ACCTGCACUTAACTAATTACATGAC Described in Methods TCGA TJOS-67 ATGACAGAUTAACTAATTACATGAC Described in Methods TCGA TJOS-97F GAATGCGTGCGATCGCGTGCATTC Described in Methods CAGCTGCATTAATGAATCGG TJOS-97R CAGCTGGCGTAATAGCGAAG Described in Methods

Supplementary Table 4. List of grnas. Target site ADE2 URA3 HIS3 HIS3_2 PDC5 ARO10 Sequence AATTGTAGAGACTATCCACA TGCAAGGGCTCCCTATCTAC GAGGCCCTAGGGGCCGTGCG ATCACACCACTGAAGACTGC TTTCACAGTCGGCGCTCTAT TTAATAACCACGTATGGCGT Supplementary Table 5. Yeast lysis protocol. Temperature ( C) Time (seconds) 65 30 8 30 65 90 97 180 8 60 65 180 97 60 65 60 80 hold