Selection strains were generated using a CRISPR-assisted knock-in method, essentially as described previously68. In brief, parent strains were transformed with the Wt Cas9 expression vector (pNA306) prior to editing, and then sgRNA plasmid (~60 ng) and gel-purified linear donor fragments (~100–300 ng) were co-delivered in a second round of transformation. Transformants were plated on SC-Leu-Ura media to maintain both plasmids, and colonies were patched onto SC-Leu-Ura and either SC-Lys or SC-Arg+Can plates to score for disruption of the LYS2 or CAN1 locus, respectively. Candidate SC-Leu-Ura patches were sequenced at their target locus by colony PCR, and sequence-verified isolates were subsequently inoculated from patch scars into YEPD broth to promote loss of the CRISPR plasmids. After overnight growth, cultures were diluted and spread on YEPD plates to obtain single colonies and replica-plated to score for plasmid loss. Strain isolates cured of both plasmids were ultimately transformed with the hEGFP sgRNA plasmid (pGG197) for use in downstream selection experiments. Single-target selection strains were generated directly from BY4741. To generate dual-target selection strains, a single-target strain that was cured of only the sgRNA plasmid was used as an intermediate for a second round of editing at the other locus. Donor templates included a single-base synonymous recode to break the PAM of the native target, which, is otherwise regenerated beyond the downstream repeat after knock-in. All donors also included ~40 bp homology arms and were PCR-amplified with synthetic linear templates and oligos listed in Supplementary Data 5; their expected sequences post amplification are listed in Supplementary Data 4. A summary of the S. cerevisiae strains used in this work is also provided in Supplementary Data 6.

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