pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae

Abstract

CRISPR-Cas9 technology is widely used for precise and specific editing of Saccharomyces cerevisiae genome to obtain marker-free engineered hosts. Targeted Double Strand Breaks (DSB) is controlled by a guide RNA (gRNA), a chimeric RNA containing a structural segment for Cas9 binding and a 20-mer guide sequence that hybridises to the genomic DNA target. Introducing the 20-mer guide sequence into gRNA expression vectors often requires complex, time-consuming and/or expensive cloning procedures. We present a new plasmid for CRISPR-Cas9 genome-editing in S. cerevisiae, pCEC-red. This tool allows i) to transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and ii) to insert the 20-mer sequence in the plasmid with high efficiency, thanks to Golden Gate Assembly and iii) a red chromoprotein-based screening to speed up the selection of correct plasmids. We tested genome-editing efficiency of pCEC-red by targeting the ADE2 gene. We chose three different 20-mer targets and designed two types of repair fragments to test pCEC-red for precision editing and for large DNA region replacement procedures. We obtained high efficiencies (close to 90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing.

Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit for Fast Engineering of Heterologous Productions in Saccharomyces cerevisiae

“…In conclusion, with this work, we remarked upon the importance of designing efficient, modular, and ready-to-use synthetic biology toolkits to accelerate the construction of the final desired cell factory and, overall, to improve yields, titers, and productivities of final strains. In recent years, there are several examples describing optimizations of already existing toolkits both for modular cloning and for genome editing …”

“…In recent years, there are several examples describing optimizations of already existing toolkits both for modular cloning 24 and for genome editing. 25 …”

Practical Approaches for the Yeast Saccharomyces cerevisiae Genome Modification

“…pCRCT encodes Cas9, tracrRNA, crRNAs, and the URA3 selectable marker [123], while pTAJAK plasmids express gRNA and contain different selectable markers [124]. pCEC-red, used for the expression of Cas9 endonuclease and gRNA, contains the marker mRFP1 (codes for the red fluorescent protein), which is replaced by gRNA when cloned using the Golden Gate Assembly method [125].…”