ArticleFEMS yeast research2023
pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.
Article in FEMS yeast research, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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4 citing papers in PubMed, 5 citations in OpenAlex.
- Challenges in elucidating ethylene glycol metabolism in Saccharomyces cerevisiae.FEMS yeast research · 2025Article
- Genomically integrated cassettes swapping: bringing modularity to the strain level in Saccharomyces cerevisiae.FEMS yeast research · 2025Article
- Practical Approaches for the YeastInternational journal of molecular sciences · 2023Review
- Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit for Fast Engineering of Heterologous Productions inACS synthetic biology · 2023Article
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4 authors at 1 institution in 1 country.
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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 are 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 to (i) transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and (ii) 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 (∼90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing.
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