Supplementary Material for StairSTEPS Manuscript

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1 Supplementary Material for StairSTEPS Manuscript S1. Supplementary Materials and Methods S1.1 Plasmid vector construction All plasmids for expression of dcas9 were derived from the pjed103 vector series acquired from AddGene catalog #46921 (Gilbert et al., 2013). All RGR plasmids were derived from the dcas9-mxi1and dcas9-vpr plasmids previously reported (Deaner and Alper, 2017). All plasmids containing an SNR52 expression cassette were cloned according to the inverse PCR/blunt-end ligation protocol previously reported by Deaner and Alper (2017). All vectors containing two SNR52 cassettes were constructed via Gibson assembly of linear SNR52- sgrna-sup4t cassettes PCR d with MD0443/MD0444 into one of the previously-constructed single SNR52 vectors digested with SpeI. Rapid cloning of TEF1-RGR-TKC27t cassettes was facilitated via the construction of a universal cloning vector. First, a 3-piece Gibson assembly of inserts TEF1 (primers MD00506/MD0507) and SpeI-sgRNA scaffold-hdv-tkc27t (primers MD0508/MD0502) into the dcas9-vpr and dcas9-mxi1 vectors linearized with SpeI. These new vectors were entitled RGR Cloning Vectors (RGR CV). To construct new RGRs, these vectors were linearized at the 5 end of the sgrna scaffold using the SpeI enzyme and then a 100 bp fragment containing a variable HH-sgRNA sequence was inserted via Gibson assembly to create the full TEF1p-HHsgRNA-HDV-TKC27t cassette. The 100 bp insert fragments were constructed by an anneal/extend PCR using two 60-bp oligos (IDT) with 20 bp overlaps at their 3 ends. To clone multiple RGRs into an RGR array, previously-constructed single RGRs (Table S4) were PCR d to create linear fragments with a specific 25 or 30 bp homology region at their

2 5 and 3 ends to allow directional cloning of RGR arrays. RGR cassettes were directionally assembled, as each subsequent RGR cassette contained a 5 homology region that was homologous to the RGR cassette positioned ahead in the array, and a 3 homology region that was homologous to the RGR cassette positioned behind in the array. Each homology region was constructed using the orthogonal sequence designer R2oDNA Designer with 50% GC content. In total, there were 5 unique homology regions used to directionally clone RGR arrays, including the 5 and 3 UTR of the RGR cistron. To create each 5 homology region for Gibson, primers with the desired 5 homology sequence and a variable binding sequence specific to the desired RGR were used. For instance, the forward primers used to position the RGR targeted to the +88 region of YFP within the cistron were MD0560 (Position 1), MD0643 (Position 2), MD0644 (Position 3), and MD0645 (Position 4). The 3 homology regions used to clone each RGR into a specific position within the cistron were produced by the following reverse primers with binding site at the 3 end of the HDV ribozyme in the RGR cassette: MD0557 (position 1), MD0591 (position 2), MD0626 (position 3), and MD0559 (position 4). All multiple RGR arrays along with the primers used for Gibson assembly are displayed in Table S5, and the flanking sequences for each position in the arrays are listed in Table S6. To swap GAL1p for TEF1p driving expression of the RGR cistron, GAL1p was PCR amplified from a p415-gal1p-cas9 plasmid commonly used in the lab using primers MD0818/MD0820. This linear product was then Gibson assembled along with a fragment PCR amplified from template TEF1-RGR-+88(YFP)/-102(TPI1p)/-178(GPD1p) (primers MD0817/MD0566) into vector RGR CV digested with SpeI/PacI.

3 S1.2 Detailed strain construction To construct the TRP1::PTPI1/PTDH3/PTEF1-yECitrine strains for flow cytometry analysis, first a PTPI1/PTDH3/PTEF1-yECitrine-CYC1t cassette was constructed in Mumberg plasmid p416. Next, 80 base pairs of homology were added to the cassette via PCR with primers JA080 and MD0060. Expression cassettes were gel extracted and then transformed into BY4741 using the traditional Gietz protocol. Transformants were selected on CSM-URA agar plates. S1.3 Quantitative PCR protocol and analysis RNA was extracted from two biological replicates for each construct using Zymolyase digestion of the yeast cell wall followed by purification using the Quick-RNA MiniPrep kit according to manufacturer s instructions (Zymo Research Corp). For qpcr experiments measuring sgrna expression, cdna was generated from the purified RNA using the Protoscript II Reverse Transcription Kit (NEB) using reverse primers specific for the sgrna scaffold and ALG9 (SI Appendix, Table 3). For qpcr experiments measuring native gene expression, cdna was generated from the purified RNA using the High Capacity cdna Reverse Transcription Kit (Applied Biosystems) with random primers following manufacturer s instructions. Primers (SI Appendix, Table 3) for qpcr were designed using the PrimerBlast tool and obtained from Integrated DNA Technologies. Quantitative PCR was performed on a ViiA7 qpcr system (Life Technologies) using SYBR Green Master Mix from Roche with ROX as a passive reference. qpcr was performed according to manufacturer s instructions with an annealing temperature of 60 C and 25 ng of total cdna product per 10 µl reaction in a 384-well optical plate. The ALG9 gene was used as a housekeeping gene, and the relative transcript levels for sgrna, NDE2,

4 CYC1, TDH1, and GPD1 were obtained by calculating the average values of the threshold cycle (Ct) between three technical replicates for each sample. Standard curves for each primer pair were constructed by serially diluting the strongest gene-overexpressing strain (for dcas9-vpr activation) or the negative control strain (for dcas9-repression) from 50 ng to 0.1 ng input cdna and calculating the slope of the semi-log plot of Ct vs. cdna input (not shown) using weighted least squares regression. For the qpcr experiment depicted in Figure S3, all parameters were kept the same as the protocol above. However, the strains used to generate cdna standard curves for each primer was the strain containing single RGR expression of sgrna +88 (RGR1) for Figures S3A, S3B, and S3D, and RGR arrays P2-P4 for figure S3C. The Pfaffl equation was used in order to factor primer efficiency into the calculations of relative transcript levels for all experiments. For some samples, one technical replicate was discarded if it did not closely match the other two replicates and the total standard deviation of the three replicates was greater than 0.1. In figure S3C, the normalized abundance of each sgrna (RGR1-4) was calculated for each RGR array (P1-P4). Next, the ratio of sgrna abundance between array positions was calculated, and these values were used to calculate average normalized sgrna abundances for each position within the array. As an example, the abundance ratio of RGR2 (sg19) at position 1 versus position 2 was calculated by dividing the average amount of RGR2 in RGR arrays P2-P4 by the average amount in array P1. This same procedure was carried out for each sgrna in the array. At the end, the average abundance ratios between positions P1/P2, P2/P3, and P3/P4 were calculated. For instance, to calculate P1/P2, the ratio of RGR2 in position 1:2 and the ratio of RGR1 in position 1:2 were averaged. Next, the abundance of sgrna at P4 was set equal to 1 and then the average sgrna abundances at each position were calculated by multiplying the proper

5 ratios. For instance, P1/P4=(P1/P2)*(P2/P3)*(P3/P4) and so on. Standard deviations of three technical replicates for each RGR array were included with each quantity and propagated at each mathematical step.

6 S2. Supplementary Figures Figure S1. Schematic depicting crosstalk between non-orthogonal dcas9 fusions. When two non-orthogonal dcas9 fusions (such as dcas9-vpr and dcas9-mxi1 from Streptococcus pyogenes) are expressed in the same cell, they will be targeted to the same genes depending on the sgrnas expressed. Thus, it is impossible to control which genes are activated and which are repressed based on the current dcas9-regulator system.

7 Figure S2. Comparison of dcas9 and dcas9-vpr for CRISPRi repression. (A) Repression of TPI1p/TDH3p-YFP using dcas9 (light) and dcas9-vpr (dark) targeted +88 bp of YFP. Flow cytometry experiment was carried out similarly to that of Figure 3. Mean YFP fluorescence of biological triplicate was calculated for each construct, and the % change in fluorescence based on a no sgrna control is reported. Error bars reported are standard deviation of the triplicate. (B) dcas9 (grey) and dcas9-vpr (black) are stepped towards the start codon using the sgrna set from Figure 1 in order to create graded repression of the TPI1 promoter.

8 Figure S3. Comparison of sgrna abundances at different RGR array positions via qpcr. sgrna abundances produced by the constructs in Figure 8A are reported here. (A) The total amount of the YFP-repressing +88 sgrna (RGR1) and the amount normalized to the total amount of sgrna is plotted as a function of RGR position within the 4x RGR array. (B) Total sgrna amount is plotted as a function of position. (C) The amount of each sgrna within the RGR array is quantified and the normalized average amounts at each array position are reported. (D) Repression of YFP for each RGR array (P1-P4) is plotted against the amount of +88 sgrna generated by each array. Both repression and sgrna amount are normalized to expression of +88 sgrna using a single RGR. sgrna abundance values and error bars in (A), (B), and (D) are reported as the average and standard deviation of biological duplicate (n=2). Results in (C) are reported as the average and standard deviation of sgrna abundances for each position within arrays P1-P4. More information on data processing for Figure S3C is reported in Section S1.3 paragraph 2.

9 S3. Supplementary Tables Table S1. List of strains used in this study Strain Genotype Parent strain TRP::PTPI1- Mat α; his3δ1; leu2δ0; met15δ0; ura3δ0; BY4741 yecitrine trp1δ::ptpi1-yecitrine-cyc1t TRP::PTDH3- Mat α; his3δ1; leu2δ0; met15δ0; ura3δ0; BY4741 yecitrine trp1δ::ptdh3- yecitrine -CYC1t TRP::PTEF1- Mat α; his3δ1; leu2δ0; met15δ0; ura3δ0; BY4741 yecitrine trp1δ::ptef1-yecitrine-cyc1t TRP::GPP1pyECitrine Mat α; his3δ1; leu2δ0; met15δ0; ura3δ0; BY4741 trp1δ::gpp1p-yecitrine-cyc1t GDTY Mat α; his3δ1; leu2δ0; met15δ0; ura3δ0; BY4741 trp1δ::pgpd-dhab-prm9t-ptef-yqhd- SPG5t References This study This study This study This study Deaner and Alper 2017

10 Table S2. List of qpcr/reverse transcription Primers used in study Purpose Target Forward Primer Reverse Primer RT/qPCR RT/qPCR qpcr qpcr qpcr qpcr qpcr qpcr qpcr qpcr qpcr sgrna Alg9 1 Alg9 2 NDE2 CYC1 TDH1 GPD1 sg13 sg19 sg20 sg21 TTCTGTCTCCGGTGAAGGTGAA ATCGTGAAATTGCAGGCAGCTT ATCGTGAAATTGCAGGCAGCTT TTTTGCGAGGACTGCTAGGT CAAGGCCGGTTCTGCTAAGA CTGTTTCCCATGACGACAAGC GGCAAGGACGTCGACCATAA AGCATCACCTTCACCTTCAC ATCCAGCAGTATTCGGTCAG ACTCTGCTATCAGGTAAGCG GTTCTTACAACGGCAGCAGT TAAGGTTGGCCATGGAACTGGCAA CATGGCAACGGCAGAAGGCAATAA CATGGCAACGGCAGAAGGCAATAA GCCAACGTCGTGTAAAAGGT AGCTTGACCAGAGTGTCTGC CAGTGGAGTCAACAGCGACA ACCGACTCTTTGGATGGCAG TAAGGTTGGCCATGGAACTGGCAA TAAGGTTGGCCATGGAACTGGCAA TAAGGTTGGCCATGGAACTGGCAA TAAGGTTGGCCATGGAACTGGCAA

11 Table S3. List of Primers used in study Name Purpose Sequence MD0170F PCR out GPP1 w/ SacI GATCGAGCTCACTATGGAATAATACAATGCACA C MD0170R PCR out GPP1 w/ XbaI GTACTCTAGATGCGATGGTTTGTATATTTGC MD0171F PCR out TPI1 w/ SacI GATCGAGCTCAAGGATGAGCCAAGAATAAGG MD0171R PCR out TPI1 w/ XbaI GTACTCTAGATTTTAGTTTATGTATGTGTTTTTTG TAGTTATAG MD0352 Inverse PCR from 3' end GATCATTTATCTTTCACTGCGG of SNR52 MD0400 Inverse PCR from 5' end of sgrna with sg11 GAAAAATATTTACGTAAGAAGTTTTAGAGCTAG AAATAGCAAGTTAAAA MD0401 Inverse PCR from 5' end of sgrna with sg12 CCATCTAATTCAACCAAAATGTTTTAGAGCTAGA AATAGCAAGTTAAAA MD0402 Inverse PCR from 5' end of sgrna with sg13 AGCATCACCTTCACCTTCACGTTTTAGAGCTAGA AATAGCAAGTTAAAA MD0403 Inverse PCR from 5' end of sgrna with sg14 ACTAAGGTTGGCCATGGAACGTTTTAGAGCTAG AAATAGCAAGTTAAAA MD0404 Inverse PCR from 5' end of sgrna with sg15 CTTTCTTGAACATAACCTTCGTTTTAGAGCTAGA AATAGCAAGTTAAAA MD0405 Inverse PCR from 5' end AAGACTGGACCATCACCAATGTTTTAGAGCTAG MD0502 MD0508 of sgrna with sg16 Amplify sgrna-hdv- TKC27t with homology to dcas9-vpr digest with SpeI Rev Fwd Amplify sg scaffold with homology to TEF with SpeI site MD0506 Fwd Amplify TEF with 5' homology to dcas9-vpr MD0507 Rev Amplify TEF with 5' homology to dcas9-vpr with SpeI site MD0509 MD0510 MD0511 MD0512 MD0519 MD0520 sg17-gibfrag1-fwd Anneal for Gibson sg17-gibfrag1-rev Anneal for Gibson sg17-gibfrag2-fwd Anneal for Gibson sg17-gibfrag2-rev Anneal for Gibson PCR out TEF-HH-sg17- HDV-TKC27 to clone into dcas9-mxi1 vector PCR out TEF-HH-sg17- HDV-TKC27 with SpeI AAATAGCAAGTTAAAA GCAGTATTGATAATGATAAACTCGAACTGATTTG AAAGATGATACTCTTTATTCC TCTAATCTAAGTTTACCCCATCGATCACTAGTTTT AGAGCTAGAAATAGCAAG CGAATTGGAGCTCACTCGGAATTCAGTCTAATAG CTTCAAAATGTTTCTACTCC ACTAGTGATCGATGGGGTAAACTTAGATTAG CAATCTAATCTAAGTTTACCCCATCGATCATTGC TTCTGATGAGTCCGTGAGGACGAAAC TAAAACAGAGAATTGTGTGATTGCTTGACGAGCT TACTCGTTTCGTCCTCACGGACT CGTCAAGCAATCACACAATTCTCTGTTTTAGAGC TAGAAATAGC ATAACGGACTAGCCTTATTTTAACTTGCTATTTCT AGCTCTAAAACAGAG GATCGAATTCATAGCTTCAAAATGTTTCTACTC GATCACTAGTTTTGAAAGATGATACTCTTTATTC C

12 MD0523 PCR of GPD1 with 5' p413 homology MD0524 PCR of GPD1 with 3' mcherry homology MD0525 PCR of mcherry with 5' GPD1 homology MD0526 PCR of mcherry with 3' PRM9 Homology MD0529 Sequencing of p413- GPD1-mCherry-PRM9 start 562 in plasmid map MD0530 Anneal/Extend Fwd gibson sg13 into RGR CV MD0531 Anneal/Extend Rev gibson sg13 into RGR CV MD0532 Anneal/Extend Fwd gibson sg11 into RGR CV MD0533 Anneal/Extend Rev gibson sg11 into RGR CV MD0534 Anneal/Extend Fwd gibson sg14 into RGR CV MD0535 Anneal/Extend Rev gibson sg14 into RGR CV MD0536 Anneal/Extend Fwd gibson sg15 into RGR CV MD0537 Anneal/Extend Rev gibson sg15 into RGR CV MD0538 Anneal/Extend Fwd gibson sg16 into RGR CV MD0539 Anneal/Extend Rev gibson sg16 into RGR CV MD0540 Anneal/Extend Fwd gibson sg18 into RGR CV MD0541 Anneal/Extend Rev gibson sg18 into RGR CV MD0546 PCR HH-sg18-HDV with Homology to RGR-sg17 digested with PacI MD0547 PCR HH-sg18-HDV with Homology to RGR-sg17 digested with PacI MD0556 Fwd PCR generate GibsonFrag1-sg17-sg18 MD0557 Rev PCR generate GibsonFrag1-sg17-sg18 MD0558 Fwd PCR generate GibsonFrag2-sg17-sg18 MD0559 Rev PCR generate GibsonFrag2-sg17-sg18 CCCTCACTAAAGGGAACAAAAGCTGGAGCTCAG CCCGAAAGAGTTATCGTTAC CTTTATATTATCAATATTTGTGTTTGTGGAGG TCCACAAACACAAATATTGATAATATAAAGATC CATGGTGAGCAAGGGC TGTGTGCTAGTGTCTCCCGTCTTCTGTGATATCCT ACTTGTACAGCTCGTCCATGC CCTATGTCTCTGGCCGATCA AAGTTTACCCCATCGATCAGATGCTCTGATGAGT GCTATTTCTAGCTCTAAAACGTGAAGGTGAAGGT GATGCTGACGAGCTTACTCGTTTCGT AAGTTTACCCCATCGATCATTTTTCCTGATGAGT GCTATTTCTAGCTCTAAAACTTCTTACGTAAATA TTTTTCGACGAGCTTACTCGTTTCGT AAGTTTACCCCATCGATCACTTAGTCTGATGAGT GCTATTTCTAGCTCTAAAACGTTCCATGGCCAAC CTTAGTGACGAGCTTACTCGTTTCGT AAGTTTACCCCATCGATCAAGAAAGCTGATGAG T GCTATTTCTAGCTCTAAAACGAAGGTTATGTTCA AGAAAGGACGAGCTTACTCGTTTCGT AAGTTTACCCCATCGATCAAGTCTTCTGATGAGT GCTATTTCTAGCTCTAAAACATTGGTGATGGTCC AGTCTTGACGAGCTTACTCGTTTCGT AAGTTTACCCCATCGATCATAGGTTCTGATGAGT GCTATTTCTAGCTCTAAAACGTCTACGTGCGAAT TAGGTTGACGAGCTTACTCGTTTCGT TCGGCATGGCGAATGGGACAGCTTTGGACTTTAA TTAGGTTCTGATGAGTCCGTGAGG TATTCCTACATAAGTAAATGAGTTTATATATTAA TAAGCTGTCCCATTCGCCATG CAATCTAATCTAAGTTTACCCCATCGATCATTGC TTCTGATGAGTCCGTG AAGCCACTCGACGCGTTGTTACAGAAAAGCTGT CCCATTCGCC TCTGTAACAACGCGTCGAGTGGCTTTAGGTTCTG ATGAGTCCG CTCTTTATTCCTACATAAGTAAATGAG

13 MD0560 MD0561 MD0562 MD0563 MD0564 MD0566 MD0590 MD0591 MD0592 MD0606 MD0607 MD0608 MD0609 MD0610 MD0611 MD0617 MD0618 MD0619 MD0620 MD0621 Fwd PCR generate GibsonFrag1-sg13-X (use w/md0557) Fwd PCR generate GIbsonFrag2-sg13-sg11 (USE W/ MD0559) Fwd PCR generate GIbsonFrag2-sg13-sg14 Fwd PCR generate GIbsonFrag2-sg13-sg15 Fwd PCR generate GIbsonFrag2-sg13-sg16 Rev PCR of RGR-sg17- TKC27 with homology to TDH3-VPR dig. SpeI Fwd PCR of TEF homology upstream of RGRs for Gibson (use with 557) Rev PCR of RGRs with homology to MD0592 Fwd PCR of HH-sg18 to generate Gibson Frag 3 (use with 559) Fwd AE for Gibson of RGR-sg19 Rev AE for Gibson of RGR-sg19 Fwd AE for Gibson of RGR-sg20 Rev AE for Gibson of RGR-sg20 Fwd AE for Gibson of RGR-sg21 Rev AE for Gibson of RGR-sg21 Fwd PCR of RGR-sg17 to clone into position 2 for RGR shift Fwd PCR of RGR-sg17 to clone into position 3 for RGR shift Fwd PCR of RGR-sg17 to clone into position 4 for RGR shift Fwd PCR of RGR-sg19 to clone into position 1 Fwd PCR of RGR-sg19 to clone into position 2 CAATCTAATCTAAGTTTACCCCATCGATCAGATG CTCTGATGAGTCCGTG TCTGTAACAACGCGTCGAGTGGCTTTTTTTCCTG ATGAGTCCG TCTGTAACAACGCGTCGAGTGGCTTCTTAGTCTG ATGAGTCCG TCTGTAACAACGCGTCGAGTGGCTTAGAAAGCT GATGAGTCCG TCTGTAACAACGCGTCGAGTGGCTTAGTCTTCTG ATGAGTCCG GCAGTATTGATAATGATAAACTCGAACTG caatctaatctaagtttaccccatcgatcac GTGGCGGAACTCTGCTGAAAATCTCTACACaaagct GTCCCATTCGC GTGTAGAGATTTTCAGCAGAGTTCCGCCACTAGG TTCTGATGAGTCCG AAGTTTACCCCATCGATCACTGGATCTGATGAGT GCTATTTCTAGCTCTAAAACCTGACCGAATACTG CTGGATGACGAGCTTACTCGTTTCGT AAGTTTACCCCATCGATCACAGAGTCTGATGAGT GCTATTTCTAGCTCTAAAACCGCTTACCTGATAG CAGAGTGACGAGCTTACTCGTTTCGT AAGTTTACCCCATCGATCAAAGAACCTGATGAGT GCTATTTCTAGCTCTAAAACACTGCTGCCGTTGT AAGAACGACGAGCTTACTCGTTTCGT TCTGTAACAACGCGTCGAGTGGCTTTTGCTTCTG ATGAGTCCGTG GTGTAGAGATTTTCAGCAGAGTTCCGCCACTTGC TTCTGATGAGTCCGTG TTGGAGCACAGTAGACCGATAGTCGTTCTCTTGC TTCTGATGAGTCCGTG CAATCTAATCTAAGTTTACCCCATCGATCACTGG ATCTGATGAGTCCGTG TCTGTAACAACGCGTCGAGTGGCTTCTGGATCTG ATGAGTCCGTG

14 MD0622 Fwd PCR of RGR-sg20 to clone into position 2 TCTGTAACAACGCGTCGAGTGGCTTCAGAGTCTG ATGAGTCCGTG MD0623 Fwd PCR of RGR-sg20 to clone into position 3 GTGTAGAGATTTTCAGCAGAGTTCCGCCACCAGA GTCTGATGAGTCCGTG MD0624 Fwd PCR of RGR-sg21 to clone into position 3 GTGTAGAGATTTTCAGCAGAGTTCCGCCACAAG AACCTGATGAGTCCGTGAG MD0625 Fwd PCR of RGR-sg21 to clone into position 4 TTGGAGCACAGTAGACCGATAGTCGTTCTCAAG AACCTGATGAGTCCGTGAG MD0626 Rev PCR to clone into position 3 GAGAACGACTATCGGTCTACTGTGCTCCAAAAA GCTGTCCCATTCGCC MD0643 Fwd PCR of RGR-sg13 to clone into position 2 for TCTGTAACAACGCGTCGAGTGGCTTGATGCTCTG ATGAGTCCGTG RGR shift MD0644 Fwd PCR of RGR-sg13 to clone into position 3 for GTGTAGAGATTTTCAGCAGAGTTCCGCCACGATG CTCTGATGAGTCCGTG RGR shift MD0645 Fwd PCR of RGR-sg13 to clone into position 4 for TTGGAGCACAGTAGACCGATAGTCGTTCTCGATG CTCTGATGAGTCCGTG RGR shift MD0656 DM1 Fwd NDE2 AAGTTTACCCCATCGATCAAGAGAGCTGATGAG T MD0657 DM1 Rev NDE2 GCTATTTCTAGCTCTAAAACCCGCAGATGCTAGA AGAGAGGACGAGCTTACTCGTTTCGT MD0658 DM2 Fwd NDE2 AAGTTTACCCCATCGATCAAACCCTCTGATGAGT MD0659 DM2 Rev NDE2 GCTATTTCTAGCTCTAAAACGTAGGCTGTAGAGA AACCCTGACGAGCTTACTCGTTTCGT MD0660 DM3 Fwd CYC1 AAGTTTACCCCATCGATCACGATGTCTGATGAGT MD0661 DM3 Rev CYC1 GCTATTTCTAGCTCTAAAACTGAATCATATTCGA CGATGTGACGAGCTTACTCGTTTCGT MD0662 DM4 Fwd CYC1 AAGTTTACCCCATCGATCATAAAGTCTGATGAGT MD0663 DM4 Rev CYC1 GCTATTTCTAGCTCTAAAACGTATGTGTCAGCAC TAAAGTGACGAGCTTACTCGTTTCGT MD0697 Fwd qpcr CYC1 CAAGGCCGGTTCTGCTAAGA MD0698 Rev qpcr CYC1 AGCTTGACCAGAGTGTCTGC MD0728 Fwd PCR of DM1 to clone into position 1 CAATCTAATCTAAGTTTACCCCATCGATCAAGAG AGCTGATGAGTCCGTG MD0729 Fwd PCR of DM2 to clone into position 1 CAATCTAATCTAAGTTTACCCCATCGATCAAACC CTCTGATGAGTCCGTG MD0730 Fwd PCR of DM3 to clone into position 4 TTGGAGCACAGTAGACCGATAGTCGTTCTCCGAT GTCTGATGAGTCCGTG MD0731 Fwd PCR of DM4 to clone into position 4 TTGGAGCACAGTAGACCGATAGTCGTTCTCTAAA GTCTGATGAGTCCGTGAGG MD0797 DM5 Target TDH1 Fwd AAGTTTACCCCATCGATCACAGTACCTGATGAGT MD0798 DM5 Target TDH1 Rev GCTATTTCTAGCTCTAAAACACACACACAAAAA ACAGTACGACGAGCTTACTCGTTTCGT

15 MD0799 DM6 Target TDH1 Fwd AAGTTTACCCCATCGATCATTATGCCTGATGAGT MD0800 DM6 Target TDH1 Rev GCTATTTCTAGCTCTAAAACTTTATCTCTAACGA TTATGCGACGAGCTTACTCGTTTCGT MD0801 DM7 Target TDH1 Fwd AAGTTTACCCCATCGATCACAGACACTGATGAGT MD0802 DM7 Target TDH1 Rev GCTATTTCTAGCTCTAAAACCACTAAATACACTC CAGACAGACGAGCTTACTCGTTTCGT MD0803 DM8 Target TDH1 Fwd AAGTTTACCCCATCGATCAGTTGATCTGATGAGT MD0804 DM8 Target TDH1 Rev GCTATTTCTAGCTCTAAAACCCACTCAAAAGACT GTTGATGACGAGCTTACTCGTTTCGT MD0805 DM9 Target GPD1 Fwd AAGTTTACCCCATCGATCAACGTTACTGATGAGT MD0806 DM9 Target GPD1 Rev GCTATTTCTAGCTCTAAAACTGAGTGAAACTGTT ACGTTAGACGAGCTTACTCGTTTCGT MD0807 DM10 Target GPD1 Fwd AAGTTTACCCCATCGATCAGATTGGCTGATGAGT MD0808 DM10 Target GPD1 Rev GCTATTTCTAGCTCTAAAACTTCAAGGTTACTGT GATTGGGACGAGCTTACTCGTTTCGT MD0809 DM11 Target GPD1 Fwd AAGTTTACCCCATCGATCACGACAACTGATGAGT MD0810 DM11 Target GPD1 Rev GCTATTTCTAGCTCTAAAACGGCATCACTCTACC CGACAAGACGAGCTTACTCGTTTCGT MD0811 DM12 Target GPD1 Fwd AAGTTTACCCCATCGATCACACTGGCTGATGAGT MD0812 DM12 Target GPD1 Rev GCTATTTCTAGCTCTAAAACAAGTCGCTCAAGAA CACTGGGACGAGCTTACTCGTTTCGT MD0817 Fwd PCR of sg13 with ACCCCATCGATCAGATGC homology to MD0816 MD0818 Fwd PCR of GAL1p to generate Gibson frag CGAATTGGAGCTCACTCGGAATTCAGTCTATAGT ACGGATTAGAAGCCGC MD0819 Rev PCR of GAL1p with fixed TSS hom. sg17 GGACTCATCAGAAGCAATGATCGATGGGGTATC CGGGGTTTTTTCTCCTTG MD0820 Rev. PCR of GAL1p with fixed TSS hom. sg13 GGACTCATCAGAGCATCTGATCGATGGGGTATCC GGGGTTTTTTCTCCTTG MD0821 Fwd PCR of DM5 to clone into Pos. 3 GTGTAGAGATTTTCAGCAGAGTTCCGCCACCAGT ACCTGATGAGTCCGTG MD0822 Fwd PCR of DM6 to clone into Pos. 3 GTGTAGAGATTTTCAGCAGAGTTCCGCCACTTAT GCCTGATGAGTCCGTG MD0823 Fwd PCR of DM7 to clone into Pos. 3 GTGTAGAGATTTTCAGCAGAGTTCCGCCACCAGA CACTGATGAGTCCGTG MD0824 Fwd PCR of DM8 to clone into Pos. 3 GTGTAGAGATTTTCAGCAGAGTTCCGCCACGTTG ATCTGATGAGTCCGTG MD0825 Fwd PCR of DM9 to clone into Pos. 2 GCTTTTCTGTAACAACGCGTCGAGTGGCTTACGT TACTGATGAGTCCGTG MD0826 Fwd PCR of DM10 to GCTTTTCTGTAACAACGCGTCGAGTGGCTTGATT MD0827 clone into Pos. 2 Fwd PCR of DM11 to clone into Pos. 2 GGCTGATGAGTCCG GCTTTTCTGTAACAACGCGTCGAGTGGCTTCGAC AACTGATGAGTCCG

16 MD0828 Fwd PCR of DM12 to clone into Pos. 2 GCTTTTCTGTAACAACGCGTCGAGTGGCTTCACT GGCTGATGAGTCCG MD0217F qpcr of NDE2 TTTTGCGAGGACTGCTAGGT MD0217R qpcr of NDE2 GCCAACGTCGTGTAAAAGGT MD0829 Fwd qpcr of GPD1 Pair 1 GGCAAGGACGTCGACCATAA MD0830 Rev qpcr of GPD1 Pair 1 ACCGACTCTTTGGATGGCAG MD0835 Fwd qpcr of TDH1 Pair CTGTTTCCCATGACGACAAGC 1 MD0836 Rev qpcr of TDH1 Pair 1 CAGTGGAGTCAACAGCGACA MD0864 fwd qpcr of sg13 (+88) AGCATCACCTTCACCTTCAC MD0893 Fwd qpcr of sg19 (NT) ATCCAGCAGTATTCGGTCAG MD0894 Fwd qpcr of sg20 (NT) ACTCTGCTATCAGGTAAGCG MD0895 Fwd qpcr of sg21 (NT) GTTCTTACAACGGCAGCAGT

17 Table S4. List of sgrna targets used in this study Name Sequence Target Binding location relative to ORF Target strand sg11 GAAAAATATTTACGTAAGAA TPI1p -120 Sense sg12 CCATCTAATTCAACCAAAAT YFP +36 Sense sg13 AGCATCACCTTCACCTTCAC YFP +88 Sense sg14 ACTAAGGTTGGCCATGGAAC YFP +159 Sense sg15 CTTTCTTGAACATAACCTTC YFP +264 Sense sg16 AAGACTGGACCATCACCAAT YFP +558 Sense sg17 AAGCAATCACACAATTCTCT TPI1p -272 Anti-sense sg18 AACCTAATTCGCACGTAGAC GPD1p -178 Anti-sense sg19 ATCCAGCAGTATTCGGTCAG None NA (RGR2) NA sg20 ACTCTGCTATCAGGTAAGCG None NA (RGR3) NA sg21 GTTCTTACAACGGCAGCAGT None NA (RGR4) NA DM1 CTCTCTTCTAGCATCTGCGG NDE2p -679 Anti-sense DM2 AGGGTTTCTCTACAGCCTAC NDE2p -156 Anti-sense DM3 ACATCGTCGAATATGATTCA CYC1p -430 Anti-sense DM4 ACTTTAGTGCTGACACATAC CYC1p -179 Anti-sense DM5 GTACTGTTTTTTGTGTGTGT TDH1p -26 Sense DM6 GCATAATCGTTAGAGATAAA TDH1 +99 Sense DM7 TGTCTGGAGTGTATTTAGTG TDH Sense DM8 ATCAACAGTCTTTTGAGTGG TDH Sense DM9 TAACGTAACAGTTTCACTCA GPD1p -119 Sense DM10 CCAATCACAGTAACCTTGAA GPD1 +99 Sense DM11 TTGTCGGGTAGAGTGATGCC GPD Sense DM12 CCAGTGTTCTTGAGCGACTT GPD Sense

18 Table S5. Primers used to construct RGR arrays RGR Array Sg11 Sg14 Sg15 Sg16 Sg17- Sg18 Sg11- Sg18 Sg18 Sg14- Sg18 Sg15- Sg18 Sg16- Sg18 Sg11- Sg18 Sg14- Sg18 Sg15- Sg18 Sg16- Sg18 Sg17- Sg19- Sg20- Sg21 Sg19- Sg17- Sg20- Sg21 Sg19- Sg20- Sg17- Sg21 Fwd Primer Frag. 1 Rev Primer Frag. 1 Fwd Primer Frag. 2 Rev Primer Frag. 2 Fwd Primer Frag. 3 Rev Primer Frag. 3 Fwd Primer Frag. 4 Rev Primer Frag. 4 MD0560 MD0557 MD0561 MD0559 NA NA NA NA MD0560 MD0557 MD0562 MD0559 NA NA NA NA MD0560 MD0557 MD0563 MD0559 NA NA NA NA MD0560 MD0557 MD0564 MD0559 NA NA NA NA MD0556 MD0557 MD0558 MD0559 NA NA NA NA MD0560 MD0557 MD0558 MD0559 NA NA NA NA MD0590 MD0557 MD0558 MD0559 NA NA NA NA MD0590 MD0557 MD0558 MD0559 NA NA NA NA MD0590 MD0557 MD0558 MD0559 NA NA NA NA MD0590 MD0557 MD0558 MD0559 NA NA NA NA MD0560 MD0557 MD0561 MD0591 MD0592 MD0559 NA NA MD0560 MD0557 MD0562 MD0591 MD0592 MD0559 NA NA MD0560 MD0557 MD0563 MD0591 MD0592 MD0559 NA NA MD0560 MD0557 MD0564 MD0591 MD0592 MD0559 NA NA MD0556 MD0557 MD0621 MD0591 MD0623 MD0626 MD0625 MD0559 MD0620 MD0557 MD0617 MD0591 MD0623 MD0626 MD0625 MD0559 MD0620 MD0557 MD0622 MD0591 MD0618 MD0626 MD0625 MD0559

19 Sg19- Sg20- Sg21- Sg17 Sg19- Sg20- Sg21 Sg19- Sg20- Sg21 Sg19- Sg20- Sg21 Sg19- Sg20- Sg21- Sg13 DM1- DM12- DM6- DM4 DM2- DM11- DM5- DM3 DM2- DM9- DM7- DM3 DM2- DM10- DM8- DM4 MD0620 MD0557 MD0622 MD0591 MD0624 MD0626 MD0619 MD0559 MD0560 MD0557 MD0621 MD0591 MD0623 MD0626 MD0625 MD0559 MD0620 MD0557 MD0643 MD0591 MD0623 MD0626 MD0625 MD0559 MD0620 MD0557 MD0622 MD0591 MD0644 MD0626 MD0625 MD0559 MD0620 MD0557 MD0622 MD0591 MD0624 MD0626 MD0645 MD0559 MD0728 MD0599 MD0828 MD0591 MD0822 MD0626 MD0731 MD0559 MD0729 MD0599 MD0827 MD0591 MD0821 MD0626 MD0730 MD0559 MD0729 MD0599 MD0825 MD0591 MD0823 MD0626 MD0730 MD0559 MD0729 MD0599 MD0826 MD0591 MD0824 MD0626 MD0731 MD0559

20 # of RGRs 1 1 Table S6. Flanking sequences for each position in RGR arrays Position 5 Flanking 3 Flanking CAATCTAATCTAAGTTTACCCCA TCGATCA AGCTTTGGACTTTAATTAATATATA AACTC CAATCTAATCTAAGTTTACCCCA TCGATCA AGCTTTTCTGTAACAACGCGTCG AGTGGCTT CAATCTAATCTAAGTTTACCCCA TCGATCA AGCTTTTCTGTAACAACGCGTCG AGTGGCTT AGCTTTGTGTAGAGATTTTCAGC AGAGTTCCGCCAC CAATCTAATCTAAGTTTACCCCA TCGATCA AGCTTTTCTGTAACAACGCGTCG AGTGGCTT AGCTTTGTGTAGAGATTTTCAGC AGAGTTCCGCCAC AGCTTTTTGGAGCACAGTAGACC GATAGTCGTTCTC AGCTTTTCTGTAACAACGCGTCGAG TGGCTT AGCTTTGGACTTTAATTAATATATA AACTC AGCTTTTCTGTAACAACGCGTCGAG TGGCTT AGCTTTGTGTAGAGATTTTCAGCAG AGTTCCGCCAC AGCTTTGGACTTTAATTAATATATA AACTC AGCTTTTCTGTAACAACGCGTCGAG TGGCTT AGCTTTGTGTAGAGATTTTCAGCAG AGTTCCGCCAC AGCTTTTTGGAGCACAGTAGACCGA TAGTCGTTCTC AGCTTTGGACTTTAATTAATATATA AACTC