EasyGuide expanded: CRISPR applications in Saccharomyces cerevisiae supported by in vivo assembly of gRNAs and oligo pool libraries

Abstract

The CRISPR technology opened new perspectives for genetically reprogramming living organisms. In particular, CRISPR has greatly impacted the molecular genetics of the yeast Saccharomyces cerevisiae, a microbial workhorse for biotechnological applications. Recently, we proposed a new CRISPR approach (EasyGuide) based on the efficient recombination of gRNAs occurring in vivo in S. cerevisiae (ACS Synth. Biol. 2022, 11, 11, 3886-3891). The EasyGuide enables to bypass gRNA pre-cloning in E. coli, thereby accelerating genome editing procedures. In this project we propose to expand the EasyGuide toolkit by including new streamlined plasmids for efficient CRISPR applications. The new vectors will support in vivo cloning of synthetic oligo pools to assemble gRNAs for multiparallel CRISPRi (interference) and CRISPRa (activation) screenings of yeast tolerance to sugarcane bagasse lignocellulosic hydrolysates. In a novel approach, remodelled EasyGuide plasmids will facilitate cloning and genome recombination of oligo pool libraries specifying mimetic phosphorylation (Ser to Asp; Tre to Asp) and dephosphorylation (Ser to Ala; Tre to Ala) amino acid substitutions in the regulatory modules of Msn2, the key transcription factor for environmental stress responses in S. cerevisiae. By assaying MSN2-modified libraries under conditions of environmental stress (i.e., high ethanol content), or enriching activated/deactivated Msn2 variants through expression of the HSP12-GFP biosensor and Fluorescence-activated cell sorting, we intend to gain insights into critical Msn2 regulatory switches, while prospecting phospho/dephosphorylation mimetic mutations capable of improving yeast fermentation under industrial conditions. Therefore, EasyGuide is envisaged as a versatile toolkit for advancing yeast production of renewable materials and fuels. (AU)