Genetic Manipulation of S. cerevisiae Using CRISPR-Cas9

Strains used in this study are listed in Table S1. All strains were derived from the S. cerevisiae BY4742 using standard genetic techniques and CRISPR-Cas9 technology (Lee et al., 2015 (link)). C-terminal mAID-3xFlag tags (cdc33-mAID-3xFlag, tif4631-mAID-3xFlag) were generated by amplifying the SG_linker-mAID-3xFlag sequence from pNTI433 with primers that included 40 bp of sequence identity with either side of the insertion, then integrated using CRISPR-Cas9 technology as described by (Brothers and Rine, 2019 (link)). C-terminal mAID-3xV5 tag (tif4632-mAID-3xV5), and P2A-ZEM tags (aro10-P2A-ZEM and pcl5-P2A-ZEM) were constructed in the same manner by amplifying from synthetic DNA gene block (Integrated DNA Technologies) PD552 and pNTI730, respectively. Gene deletions were generated using one-step replacement with marker cassettes, more specifically by PCR amplification of the hygromycin resistance cassette from pNTI730 with flanking sequence homology to the 5’ and 3’ ends of target gene coding sequence (Goldstein and McCusker, 1999 (link); Hentges et al., 2005 (link)). PCL5-ZEM reporters and dCas9-TetR were integrated using NotI-linearized vectors from the EasyClone 2.0 toolkit for yeast genomic integration (Stovicek et al., 2015 (link)). Plasmids and primers used in strain construction are listed in Table S2 and S3, respectively.

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Diamond P.D., McGlincy N.J, & Ingolia N.T. (2023). Dysregulation of amino acid metabolism upon rapid depletion of cap-binding protein eIF4E. bioRxiv.

Publication Preprint 2023

synthetic DNA gene block

Brothers Coding sequence Crispr Gene Gene deletions Genomic Hygromycin Plasmids Primers Strain Synthetic gene Vectors Yeast

Corresponding Organization : QB3

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Variable analysis

independent variables

Genetic modifications (e.g., C-terminal mAID-3xFlag tags, C-terminal mAID-3xV5 tag, P2A-ZEM tags, gene deletions, dCas9-TetR integration)

dependent variables

Not explicitly mentioned

control variables

The S. cerevisiae BY4742 strain was used as the parent strain for all genetic modifications

controls

No positive or negative controls were explicitly mentioned in the input protocol

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