Mutants with inactivation of lncRNAs (transcr_20548 in BMA64-1A, transcr_10027 in BY4742, and transcr_3536 in SEY6210) and coding genes (CTA1 and IXR1 in BMA64-1A) were generated using CRISPR–Cas9. The genomic target regions were inserted into the pMEL16 plasmid (Addgene 107922) via PCR. Competent yeast cells were transformed with the modified pMEL16 plasmid, p414-TEF1p-Cas9-CYC1t plasmid (Addgene 43802), and the repair DNA. Recovered cells were placed on drop-out His- plus G418 medium, followed by incubation. Mutants were confirmed by colony PCR and sequencing. The population rebound after EtOH stress relief was assessed for mutants and wild-type strains, and spot tests were performed to assess the highest EtOH tolerance level of each mutant (Supplementary Table S19).
Wolf I.R., Marques L.F., de Almeida L.F., Lázari L.C., de Moraes L.N., Cardoso L.H., Alves C.C., Nakajima R.T., Schnepper A.P., Golim M.D., Cataldi T.R., Nijland J.G., Pinto C.M., Fioretto M.N., Almeida R.O., Driessen A.J., Simōes R.P., Labate M.V., Grotto R.M., Labate C.A., Fernandes Junior A., Justulin L.A., Coan R.L., Ramos É., Furtado F.B., Martins C, & Valente G.T. (2023). Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae. International Journal of Molecular Sciences, 24(6), 5646.
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