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.

Maestroni, L., Butti, P., Senatore, V., Branduardi, P. (2023). pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. FEMS YEAST RESEARCH, 23 [10.1093/femsyr/foad002].

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

Letizia Maestroni
Co-primo
;
Pietro Butti
Co-primo
;
Vittorio Giorgio Senatore
Co-primo
;
Paola Branduardi
2023

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.
Articolo in rivista - Articolo scientifico
ADE2 deletion; Saccharomyces cerevisiae ; CRISPR-Cas9; genome editing; guide RNA; new plasmid;
English
14-gen-2023
2023
23
foad002
open
Maestroni, L., Butti, P., Senatore, V., Branduardi, P. (2023). pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. FEMS YEAST RESEARCH, 23 [10.1093/femsyr/foad002].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/402342
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