Expanding the genome editing toolbox of Saccharomyces cerevisiae with the endonuclease ErCas12a

Abstract ErCas12a is a class 2 type V CRISPR–Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing...

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Bibliographic Details
Published in:FEMS yeast research 2023-01, Vol.23
Main Authors: Bennis, Nicole X, Anderson, Jonah P, Kok, Siebe M C, Daran, Jean-Marc G
Format: Article
Language:English
Subjects:
ADE2 gene
CRISPR
CRISPR-Cas Systems
Endonuclease
Endonucleases - genetics
Gene Editing
Genome editing
Genomes
Genomics
Nuclease
Protocol
RNA - metabolism
RNA processing
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Toxicity
Yeast
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Summary:Abstract ErCas12a is a class 2 type V CRISPR–Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces cerevisiae. The optimal design parameters for ErCas12a editing in S. cerevisiae were defined as a 21-nt spacer flanked by 19 nt direct repeats expressed from either RNApolII or III promoters, achieving near 100% editing efficiencies in commonly targeted genomic locations. To be able to transfer the ErCas12a genome editing tool to different strain lineages, a transportable platform plasmid was constructed and evaluated for its genome editing efficiency. Using an identical crRNA expression design, the transportable ErCas12a genome editing tool showed lower efficiency when targeting the ADE2 gene. In contrast to genomic Ercas12a expression, episomal expression of Ercas12a decreases maximum specific growth rate on glucose, indicating ErCas12a toxicity at high expression levels. Moreover, ErCas12a processed a multispacer crRNA array using the RNA self-processing capability, which allowed for simultaneous editing of multiple chromosomal locations. ErCas12a is established as a valuable addition to the genetic toolbox for S. cerevisiae. This study established ErCas12a, CRISPR-endonuclease that possesses RNA self-processing capability and the advantage of lacking reach-through royalties typical of Cas-nucleases, as a valuable addition to the S. cerevisiae genetic toolbox.
ISSN:1567-1356
1567-1364
1567-1364
DOI:10.1093/femsyr/foad043