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CRISPR interference (CRISPRi) is a powerful tool to study cellular physiology under different growth conditions, and this technology provides a means for screening changed expression of essential genes. In this study, a  Saccharomyces cerevisiae  CRISPRi library was screened for growth in medium supplemented with acetic acid. Acetic acid is a growth inhibitor challenging the use of yeast for the industrial conversion of lignocellulosic biomasses. Tolerance to acetic acid that is released during biomass hydrolysis is crucial for cell factories to be used in biorefineries. The CRISPRi library screened consists of &gt;9,000 strains, where &gt;98% of all essential and respiratory growth-essential genes were targeted with multiple guide RNAs (gRNAs). The screen was performed using the high-throughput, high-resolution Scan-o-matic platform, where each strain is analyzed separately. Our study identified that CRISPRi targeting of genes involved in vesicle formation or organelle transport processes led to severe growth inhibition during acetic acid stress, emphasizing the importance of these intracellular membrane structures in maintaining cell vitality. In contrast, strains in which genes encoding subunits of the 19S regulatory particle of the 26S proteasome were downregulated had increased tolerance to acetic acid, which we hypothesize is due to ATP salvage through an increased abundance of the 20S core particle that performs ATP-independent protein degradation. This is the first study where high-resolution CRISPRi library screening paves the way to understanding and bioengineering the robustness of yeast against acetic acid stress.   IMPORTANCE  Acetic acid is inhibitory to the growth of the yeast  Saccharomyces cerevisiae , causing ATP starvation and oxidative stress, which leads to the suboptimal production of fuels and chemicals from lignocellulosic biomass. In this study, where each strain of a CRISPRi library was characterized individually, many essential and respiratory growth-essential genes that regulate tolerance to acetic acid were identified, providing a new understanding of the stress response of yeast and new targets for the bioengineering of industrial yeast. Our findings on the fine-tuning of the expression of proteasomal genes leading to increased tolerance to acetic acid suggest that this could be a novel strategy for increasing stress tolerance, leading to improved strains for the production of biobased chemicals.

A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae