Plasmids were constructed by standard molecular biology techniques as described below and verified by Sanger sequencing (Additional File 8: Table S8). Restriction enzymes were obtained from NEB and high-fidelity (HF) variants were used when available. Q5 polymerase (NEB M0491S) was used for PCR, assembly reactions were carried out using Gibson Assembly Master Mix (NEB E2611L).
pNTI647 was generated by amplifying the adjacent dCas9-Mxi and TetR expression cassettes from pNTI601 (pRS416-dCas9-Mxi1 + TetR + pRPR1(TetO)-NotI-gRNA, Addgene #73796)  using primers NM721 and NM734 (Additional file 8: Table S8). This insert was assembled into pCfB2225 (AddGene #67553), an “EasyClone 2.0” vector for KanMX-marked integration into the XII-2 safe harbor location .
pNTI661 was generated in several steps from pNTI601. The URA3 marker was replaced by the K. lactis LEU2 marker from pUG73  by amplifying this marker using primers NI-993 and NI-994 (Additional file 8: Table S8), as well as amplifying a backbone fragment of pNTI601 using primers NI-995 and NI-996 (Additional file 8: Table S8), and assembling these back into pNTI601 digested with SpeI and KpnI. Primers KS524 and KS525 (Additional file 8: Table S8) were used to amplify the region of the vector excluding dCas9-Mxi1 and TetR, which was recircularized by Gibson assembly. The barcode site was introduced by amplifying the guide RNA expression cassette with NI-1019 and NI-1020 and re-ligating the resulting product back into the vector after a SacI/SpeI digestion of both vector and PCR amplicon. Finally, the NotI site for guide RNA cloning was replaced with a BamHI-HindIII cassette by digesting the vector with NotI and performing Gibson assembly with the NI-1030 oligonucleotide.
pNTI698 was generated by amplifying the HIS3, MET17, and URA3 genes from pHLUMv2 (AddGene #64166)  using p698Fwd and p698Rev primers (Additional file 8: Table S8). This insert was assembled into pCfB2223 (AddGene #67544), an “EasyClone 2.0” vector for KanMX-marked integration into the X-3 safe harbor location , digested with EcoNI. Note that the KanMX marker is disrupted by the HIS3-MET17-URA3 cassette and the plasmid no longer confers resistance.
Yeast were derived from S. cerevisiae strain BY4741 (ThermoFisher), a haploid MATa his3Δ1 leu2Δ0 LYS2 met15Δ ura3Δ0 derivative of S288c.
NIY416 was derived from BY4741 by transformation with integrating plasmid pNTI647 digested with NotI, followed by selection for kanamycin resistance.
NIY425 was derived from NIY416 by transformation with integrating plasmid pNTI698 digested with NotI, followed by selection for Ura and Met prototrophy.
Minimal media was prepared using 67. g / l yeast nitrogen base with ammonium sulfate and without amino acids (BD 291920) and 200. g / l dextrose (Fisher D16–500). Synthetic complete drop-out media minus leucine (SCD -Leu) was prepared using 67. g / l yeast nitrogen base with ammonium sulfate and without amino acids, 1.62 g / l synthetic drop-out mix minus leucine (US Bio D9626), and 200. g / l dextrose.
High-efficiency yeast transformations were carried out by growing yeast cultures overnight at 30 °C with shaking and diluting these cultures to prepare fresh dilution cultures at an OD600 of 0.05. Dilution cultures were grown at 30 °C with shaking until they reached an OD600 of 0.5 and then 20 ml of culture was taken for each transformation. Cells were pelleted by centrifugation at 3000×g for 10 min and the supernatant was decanted. Cells were resuspended in 10. ml sterile deionized water and pelleted again by centrifugation at 3000×g for 5 min, the supernatant was decanted, and any residual liquid was removed with a pipettor. Cells were then resuspended in 1.0 ml lithium acetate 100 mM, transferred to a microcentrifuge tube, and pelleted by centrifugation at 10,000×g for 10 s. Supernatant was removed by aspiration and cells were resuspended in 1.0 ml lithium acetate 100 mM and pelleted again at 10,000×g for 10 s. Supernatant was removed by aspiration, and 240 μl of 50% w/v polyethylene glycol was layered gently on top of cells, followed by 20. μl of freshly boiled salmon sperm DNA 10 mg / ml (Invitrogen 15,632,011), 36. μl lithium acetate 1.0 M, and 64. μl plasmid DNA. The microcentrifuge tubes were then vortexed vigorously to resuspend cells and incubated for 20 min in a 42 °C water bath, vortexing once during the incubation to maintain cells in suspension. Following this incubation, cells were pelleted by centrifugation at 10,000×g for 10 s and the transformation mixture was removed with a pipettor. Cells were resuspended in 1 ml sterile deionized water, pelleted by centrifugation at 10,000×g for 10 s, and the water was removed with a pipettor. Finally, cells were resuspended in 1.0 ml sterile deionized water per transformation.
Guide library design
External data sets
Yeast genome sequence (R64–1-1, sacCer3)  and CDS annotations  were downloaded from the UCSC genome browser, and yeast gene information was downloaded directly from the Saccharomyces Genome Database . Transcript isoform data was obtained from Pelechano et al. . ATAC-seq data was obtained from from Schep et al. , GEO accession GSE66386.
All major transcript isoforms (mTIFs) from Pelechano et al.  annotated to cover one intact ORF were considered for gene annotation. Considering the set of mTIFs for a gene, the modal (highest read count) transcription start site (TSS) was chosen as the representative transcription start site for the gene. When no transcript was annotated for the ORF, the annotated CDS was used for guide design and target prediction.
All possible guides were identified by searching for GG dinucleotides, representing the Cas9 protospacer adjacent motif (PAM) in the yeast genome sequence. Guide site uniqueness was assessed by aligning each target sequence (20 base protospacer followed by “NGG” PAM) against the yeast genome reference using Bowtie2 . Target sequences with multiple perfect genomic alignments were considered non-unique. Guides were associated with gene TSSes when the center of the target sequence fell between − 220 and + 20 nucleotides relative to the TSS. Guides were associated with CDS genes when the center of the sequence fell between − 350 and 0 nucleotides relative to the CDS. Guides were considered specific when these targeting rules associated the guide with only one single target gene. Target accessibility was determined by averaging ATAC-Seq accessibility, ranging from 0.0 for inaccessible to 1.0 for fully accessible, across all nucleotide positions in the target sequence in two replicate ATAC-Seq data sets. When no data was available, a value of 0.0 was used.
Guides were prioritized by first preferring unique guides, and then specific guides, and finally by greater ATAC-Seq accessibility. For each TSS-annotated gene, the highest-scoring guides were chosen for three zones spanning [− 220, − 141], [− 140, − 61], and [− 60, + 20] nucleotides relative to the TSS. For each CDS-annotated gene, the highest-scoring guides were chosen for four zones spanning [− 350, − 271], [− 270, − 191], [− 190, − 111], and [− 110, − 30] nucleotides relative to the start of the CDS. Additional guides were chosen, highest score first, until ten guides were chosen or all possible guides in the targeting region were exhausted.
Barcoded guide expression library
Guide library construction
The guide RNA expression vector pNTI661 was digested by taking 3.0 μg plasmid in a 75. μl reaction with 1x final concentration CutSmart buffer (NEB B7204S) with 60 U BamHI-HF (NEB R3136L) and 60 U HindIII-HF (NEB R3104S), incubated for 1 h at 37 °C, and then purified with a DNA Clean & Concentrator (Zymo D4013). The guide RNA oligonucleotide library was amplified using Q5 polymerase (NEB M0491S) according to the manufacturers instructions, using 100 pg guide oligonucleotide pool (CustomArray, Inc.) as a template and oligonucleotides NM636 and NM637 (Additional file 8: Table S8) for amplification, with 15 cycles of amplification using 10 s denaturation, 15 s annealing at 58 °C, and 15 s extension. Amplified guide RNAs were cloned in a 100 μl assembly reaction with 1.0 μg linearized pNTI661 and 1.7 μl guide RNA PCR using 2 × NEBuilder HiFi DNA Assembly Master Mix (NEB E2621L), which was incubated for 1 h at 50 °C and then purified with a DNA Clean & Concentrator with final elution into 10. μl. Purified DNA was used to transform high efficiency competent 10-beta E. coli (NEB C3019H), using 2.5 μl purified DNA per reaction in four independent transformations of 50 μl competent cells. Following transformation, transformations were pooled into 100 ml LB Carb liquid media and grown with vigorous shaking until reaching an OD600 of 3. Plasmid DNA was extracted with a QIAGEN Plasmid Midi Kit (QIAGEN 12143).
The guide expression library was digested again with BamHI-HF along with exonucleases in order to digest and degrade the majority of the guide-free plasmids. A 50 μl digestion reaction was prepared using 2 μg plasmid DNA in 1x final concentration CutSmart buffer with 20 U BamHI-HF, 5 U lambda exonuclease (NEB M0262S), and 20 U E. coli exonuclease I (NEB 0293S). Digestion was carried out for 1 h at 37 °C, followed by heat inactivation for 20 min at 80 °C. DNA was then purified using a Zymo DNA Clean & Concentrator column, with elution into 20. μl. The library was then linearized for barcode assembly in a 50 μl digestion reaction using 18. μl of eluted DNA from the previous digestion in 1x final concentration CutSmart buffer with 20 U SphI-HF (NEB R3182S). Digestion was carried out for 1 h at 37 °C, and DNA was purified again using a Zymo DNA Clean & Concentrator column.
Random nucleotide barcodes with embedded T7 RNA polymerase promoters were generated by PCR amplification from 1.0 μl NI-1026 oligonucleotide using NI-1027 and NI-1041 oligonucleotide primers (Additional file 8: Table S8). A 50 μl PCR using Q5 polymerase (NEB M0491S) according to the manufacturers instructions, with 15 cycles of amplification using 5 s denaturation, 10 s annealing at 65 °C, and 5 s extension. Product was purified using a DNA Clean & Concentrator column. Amplified barcodes were introduced in a 100 μl NEBuilder HiFi Assembly reaction containing 1 μg linearized guide library and 110 ng purified barcode PCR. DNA was purified using a DNA Clean & Concentrator column with final elution into 10 μl. Purified DNA was used to transform high efficiency competent 10-beta E. coli, using 2.5 μl purified DNA per reaction in four independent transformations of 50 μl competent cells. Following transformation, transformations were pooled into a single, 4.0 ml pool. Dilutions were plated on LB Carb agar plates to assess transformation efficiency, and 55% of the transformation was used to inoculate a 50 ml LB Carb culture while 22, 8, 6, and 4% were used to inoculate four separate 25 ml LB Carb cultures. Higher-inoculum 55 and 22% cultures were grown at 26 °C overnight, while lower-inoculum 8, 6, and 4% cultures were grown at 30 °C overnight. Based on the estimated yield of ~ 1.1 M transformants, the 22% culture was selected. DNA was isolated using a QIAGEN Plasmid Mini kit to produce the barcoded guide expression library.
Comparative barcode amplification
Guide library transformation and yeast growth
BY4741 was transformed with barcoded guide expression library in one high-efficiency transformation of ~ 100 M cells using 64 μl of plasmid DNA at 100 ng / μl. Dilutions were plated on SCD -Leu agar plates in order to estimate the transformation efficiency, indicating a yield of ~ 330,000 independent transformants. The rest of the transformation was used to inoculate 100 ml of SCD -Leu media and grown for ~ 24 h at 30 °C with shaking, at which point the OD600 increased roughly 4-fold, to 0.82. A new 100 ml SCD -Leu culture was inoculated with 400 μl of this culture and growth at 30 °C with shaking was continued overnight to yield a final OD600 of 1.7. Four aliquots of 25 ml each were taken for yeast plasmid DNA extractions. Yeast were pelleted by centrifugation for 10 min at 3100×g, and media was discarded. Cells were resuspended in 1.0 ml sterile deionized water, pelleted 10,000×g for 30 s, and water was removed by aspiration. Washed yeast pellets were stored at − 80 °C.
Linear amplification by in vitro transcription
Half of one plasmid extraction was used to prepare a 25 μl digestion in 1x final concentration CutSmart buffer with 20 U XhoI (NEB R0146L) and incubated 1 h at 37 °C. DNA was purified using a DNA Clean & Concentrator column with elution into 20. μl, and 18 μl of purified DNA was used as template in a 30 μl HiScribe T7 Quick High Yield RNA Synthesis reaction (NEB E2050S) following the protocol for short templates and incubated overnight at 37 °C. Template was degraded by adding 20 μl water followed by 4 U DNase I and continuing incubation for 15 min at 37 °C and RNA was then purified using an RNA Clean & Concentrator, with final elution into 15. μl. Purified RNA was assessed using a High Sensitivity RNA ScreenTape with an Agilent TapeStation 2200. Reverse transcription was carried out using 10 ng of purified RNA in a reaction with ProtoScript II (NEB M0368S) using 2.0 pmol NI-1032 as a gene-specific primer (Additional file 8: Table S8). Primer and template were denatured 5 min at 65 °C, kept on ice to prepare reactions, and then incubated 1 h at 42 °C followed by heat inactivation at 65 °C for 20 min. A 50 μl PCR reaction using Q5 was prepared using 5.0 μl RT product as a template without further purification, along with NEBNext Multiplex Oligos for Illumina (NEB E7600S) as primers, and amplified for 7 cycles using 5 s denaturation, 10 s annealing at 65 °C, and 10 s extension. PCR products were purified using AMpure XP beads according to the manufacturer’s instructions, using a 2 beads: 1 PCR ratio and final elution in 20. μl Tris•Cl 10 mM, pH 8.0. Products were validated using a High Sensitivity D1000 ScreenTape on an Agilent TapeStation 2200, pooled, and analyzed by 50 base single-read deep sequencing on an Illumina HiSeq with 10% phiX control. Note that the first 25 bases comprise high-diversity barcode libraries whereas the subsequent bases are monotemplate.
Exponential PCR amplification
First-round PCR was performed using Q5 polymerase, 10% of extracted yeast plasmid DNA as a template, and primers NI-956 and NI-1032 (Additional file 8: Table S8), and amplified for 16 cycles using 10 s denaturation, 15 s annealing at 65 °C, and 10 s extension. PCR products were purified using AMpure XP beads according to the manufacturer’s instructions, using a 2 beads: 1 PCR ratio and final elution in 20. μl Tris•Cl 10 mM, pH 8.0. Second-round PCR was performed exactly as described for linear amplification by in vitro transcription, except that 1.0 μl of purified first-round PCR product was used as a template. PCR libraries were validated, pooled, and sequenced in parallel with linear amplification libraries.
Barcode sequencing data was analyzed by trimming the 3′ adapter sequence “GCATGCGTGAAGTGGCGCGCCTGATA” using Cutadapt, discarding all sequences that either lacked a linker or contained a barcode sequence less than 10 nucleotides long. Barcodes were tabulated using a custom tool, “bc-count”, that collapses single-nucleotide mismatches. Barcode counts were collated across all four libraries and filtered to remove barcodes that occurred in only one library or had fewer than 33 reads total across all 4 libraries. Barcodes were also filtered to remove sequences containing XhoI sites. Barcode counts were plotted, and DESeq2 was used to estimate read count-dispersion relationships from barcode count tables.
Sequencing library construction
First-round PCR was carried out in 50 μl using Q5 polymerase with 100 ng barcoded guide library as template and primers NI-1038 and NI-956 (Additional file 8: Table S8), and 12 cycles of amplification were performed using 10 s denaturation, 15 s annealing at 67 °C, and 20 s extension. PCR products were purified using AMpure XP beads at an 0.8 beads: 1 PCR ratio and final elution in 15. μl Tris•Cl 10 mM, pH 8.0. Second-round PCR was performed with 1.0 μl of first-round PCR as template and primers NI-798 and NI-826 (Additional file 8: Table S8), and 15 cycles of amplification were performed using 10 s denaturation, 15 s annealing at 65 °C, and 20 s extension. PCR products were again purified using AMpure XP beads and validated using a High Sensitivity D1000 ScreenTape on an Agilent TapeStation 2200 prior to 150 base paired-end sequencing on an Illumina MiSeq. PhiX control DNA was mixed to account for monotemplate regions of the library. Barcode sequencing data is available under accession SRR10356224.
Sequencing data analysis
Barcodes in R1 reads were trimmed to remove the 3′ adapter sequence “GCATGCGTGAAGTGGCGCGCCTGATAGCTCGTTTAAACTG” and read pairs lacking this adapter in the R1 read, or reads with residual barcodes less than 12 nucleotides long, were discarded. Trimmed barcodes were collapsed to combine barcodes with single-nucleotide mismatches using the custom “bc-seqs” program, and guide sequences in R2 were then trimmed to remove the 5′ adapter “CGAAAC” and the 3′ adapter “AAGTTAAAAT”, leaving 20 bases of constant sequence on each side of the variable 20 nucleotide guide sequence. Read pairs where less than 20 nucleotides of residual guide sequence remained were discarded. Remaining guide sequences were aligned against a library of guide sequences using bowtie2. These alignments were used to compute barcode assignments using the custom “bc-grna” program. This tool grouped all guide alignments associated with the same barcode sequence, discarded sequences with low-quality (Q < 30) bases, and then eliminated all barcodes that lacked at least 3 high-quality guide reads. Barcodes are assigned to guides when they are supported by at least 3 high-quality reads, at least 90% of these reads align to the same guide sequence and the majority alignment to that guide has no mismatches, insertions, or deletions. Barcodes where < 90% of all reads aligned to a single majority guide were considered heterogeneous and discarded. Barcodes where the majority alignment contained mismatches, insertions, or deletions were considered defective guides. The number of barcodes in each of these categories is tabulated in the “grna-assign-barcode-fates.txt” file and the high-quality barcode-to-guide assignments are given in the “grna-assign-barcode-grna-good.txt” file.
Guide library transformation
Guide RNA library was transformed into NIY425 as described in “High-efficiency transformations.” Three independent transformations were pooled and used to inoculate a turbidostat  containing ~ 200 ml SCD -Leu media at an initial OD600 of 0.1. The culture was maintained for ~ 24 h at a target OD600 of 0.5, at 30 °C with continuous aeration and stirring. A 40 ml culture was combined with 40 ml fresh, pre-warmed SCD -Leu media and grown in batch culture at 30 °C with shaking for 4.5 h, reaching an OD600 of 2.0. Cells were pelleted by centrifugation for 10 min at 3100×g, room temperature and media was discarded. Cells were resuspended in 8.0 ml sterile deionized water and split into 8 aliquots of 1.0 ml. Cells were pelleted 10,000×g for 30 s and water was removed by aspiration. Cells were resuspended in 0.80 ml sterile 30% glycerol in deionized water, flash frozen in liquid nitrogen, and stored at − 80 °C.
Two independent turbidostats  each containing ~ 200 ml minimal media were inoculated with aliquots of the guide library transformant pool, yielding an initial OD600 of 0.1. Turbidostats were grown at 30 °C with continuous aeration and stirring, with a target OD600 of 0.5. After ~ 46 h, a 50 ml sample was withdrawn from each turbidostat and processed as described for “Guide library transformation and yeast growth” in “Comparative barcode amplification. Turbidostat media was then replaced with minimal media containing 250 μg / l anhydrotetracycline and growth was continued, with additional 50 ml samples taken at ~ 72 h, ~ 90 h, and ~ 107 h.
Barcode abundance library construction
Plasmid DNA was extracted from frozen yeast pellets. Barcodes were amplified and sequenced as described above for “Linear amplification by in vitro transcription” in “Comparative barcode amplification,” except that 1–10 ng of in vitro transcription product was used as a reverse transcription template, and 12 cycles of PCR amplification were carried out in the final step of library generation.
Sequencing data analysis
Barcode abundance was tabulated as described above for “Comparative barcode amplification” and barcodes were matched to guides using the results of “Barcode-to-guide assignment.”
Fitness effect analyses
Barcodes were filtered to eliminate entries that did not have at least 64 reads tabulated for the pre-induction sample in at least one replicate culture. These filtered barcode counts were then analyzed using DESeq2 with the model counts ~ gens + culture, where gens was a numerical factor that was 0.0 for pre-induction samples and then 3.75, 7.5, and 11.25 for the three post-induction timepoints, and culture was a discrete factor for the two replicate cultures. The gens parameter from this linear model was taken as an estimate of the selective coefficient per population doubling for each barcode. Guide-level analysis was performed by taking the weighted mean of the estimate for each individual barcode, using the standard error estimate to compute 1/Var weights for each barcode. Fitness effect distributions were calculated by first filtering for genes with unambiguous guide targeting, where TSS data was available and no guide RNA had an alternate target gene identified by our approach. A list of essential genes was downloaded from the Saccharomyces genome deletion project [1, 2].
Guide efficacy analysis
Efficacy models were fitted using 1967 guides against essential genes with unambiguous targeting and fitness effects derived from more than one barcode. The offset between the guide target and the transcription start site was calculated based on the center of the 23 nucleotide target sequence. The relationship between fitness effect and guide-to-TSS offset was modeled with a local regression (α = 0.25) across the − 220 to + 20 range used for guide selection. Accessibility data was derived from Oberbeckmann et al. ODM-Seq data , using the lowest occupancy value in a 33 nucleotide window including the full target sequence and 5 flanking nucleotides on each side. The fitness threshold for active guides, s < − 0.38 was defined according to the 5th percentile of all negative controls. Logistic regression against activity classification was performed using the model active ~ OffsetPred + ODM + nt01 + … + nt20, where OffsetPred was the predicted value from the local regression of guide position, ODM was the ODM-Seq accessibility data, and nt01 through nt20 were 20 discrete factors representing the variable guide sequence. Alternative models excluded the ODM variable or the 20 sequence factors, included ATAC-Seq data from Schep et al.  used in guide design, or used two distinct, strand-specific local regressions for OffsetPred. Models (local regression and logistic regression together) were tested by k-fold cross-validation with k = 10, and the final model was generated using all guides. This final model was used to score 3480 guides (1491 active, i.e., log2 s < − 0.38) against essential genes that had been held out of the model development because they targeted divergent promoters or had just one barcode quantified.