CRISPR/Cas9-RNAi system for combinatorial metabolic engineering of Saccharomyces cerevisiae

Kanchana Rueksomtawin Kildegaard, Larissa Ribeiro Ramos Tramontin, Ksenia Chekina, Mingji Li, Tobias Justus Goedecke, Mette Kristensen, Irina Borodina*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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The yeast Saccharomyces cerevisiaeis 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, invivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multi-expression 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 offuture cell factories.
Original languageEnglish
Issue number5
Pages (from-to)237-247
Publication statusPublished - 2019


  • CRISPR/Cas9
  • RNA interference
  • Saccharomyces cerevisiae
  • cis,cis-muconic acid
  • genome editing
  • metabolic engineering


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