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EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9
Author(s) -
JessopFabre Mathew M,
Jakočiūnas Tadas,
Stovicek Vratislav,
Dai Zongjie,
Jensen Michael K,
Keasling Jay D,
Borodina Irina
Publication year - 2016
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201600147
Subject(s) - cas9 , crispr , saccharomyces cerevisiae , biology , genome editing , computational biology , genome , transformation (genetics) , guide rna , gene , genome engineering , genetics , metabolic engineering , vector (molecular biology) , recombinant dna
Abstract Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker‐free overexpression cassettes in the genome of S. cerevisiae , we have developed a vector toolkit EasyClone‐MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113‐7D) and a diploid industrial strain (Ethanol Red) for production of 3‐hydroxypropionic acid, where we tested three different acetyl‐CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone‐MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.