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CRISPR/Cas9‐RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae
Author(s) -
Kildegaard Kanchana Rueksomtawin,
Tramontin Larissa Ribeiro Ramos,
Chekina Ksenia,
Li Mingji,
Goedecke Tobias Justus,
Kristensen Mette,
Borodina Irina
Publication year - 2019
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.3390
Subject(s) - crispr , saccharomyces cerevisiae , metabolic engineering , cas9 , biology , computational biology , homologous recombination , genome editing , crispr interference , yeast , rna interference , genome engineering , synthetic biology , rna , biochemical engineering , microbiology and biotechnology , genetics , gene , engineering
The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high‐performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double‐strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis , cis ‐muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.