EasyGuide Plasmids Support in Vivo Assembly of gRNAs for CRISPR/Cas9 Applications in Saccharomyces cerevisiae

EasyGuide Plasmids Support in Vivo Assembly of gRNAs for CRISPR/Cas9 Applications in Saccharomyces cerevisiae

Most CRISPR/Cas9 applications in yeast rely on a plasmid-based expression of Cas9 and its guide RNA (gRNA) containing a 20-nucleotides (nts) spacer tailored to each genomic target. The lengthy assembly of this customized gRNA requires at least 3–5 days for its precloning in Escherichia coli, purific...

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Bibliographic Details
Published in:ACS synthetic biology Vol. 11; no. 11; pp. 3886 - 3891
Main Authors: Jacobus, Ana P., Barreto, Joneclei A., de Bem, Lucas S., Menegon, Yasmine A., Fier, Ícaro, Bueno, João G. R., dos Santos, Leandro V., Gross, Jeferson
Format: Journal Article
Language:English
Published: United States American Chemical Society 18-11-2022
Subjects:
CRISPR-Cas Systems - genetics
Escherichia coli - genetics
Escherichia coli - metabolism
Gene Editing - methods
Plasmids - genetics
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Guide, CRISPR-Cas Systems - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
genome editing
in vivo cloning
EasyGuide plasmids
CRISPR/Cas9
Saccharomyces cerevisiae
gRNA cloning
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Summary:Most CRISPR/Cas9 applications in yeast rely on a plasmid-based expression of Cas9 and its guide RNA (gRNA) containing a 20-nucleotides (nts) spacer tailored to each genomic target. The lengthy assembly of this customized gRNA requires at least 3–5 days for its precloning in Escherichia coli, purification, validation, and cotransformation with Cas9 into a yeast strain. Here, we constructed a series of 12 EasyGuide plasmids to simplify CRISPR/Cas9 applications in Saccharomyces cerevisiae. The new vectors provide templates for generating PCR fragments that can assemble up to six functional gRNAs directly into yeasts via homologous recombination between the 20-nts spacers. By dispensing precloning in E. coli, yeast in vivo gRNA assembly significantly reduces the CRISPR/Cas9 experimental workload. A highly efficient yeast genome editing procedure, involving PCR amplification of gRNAs and donors, followed by their transformation into a Cas9-expressing strain, can be easily accomplished through a quick protocol.
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ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.2c00348