CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

Klaudia Ciurkot, Brenda Vonk, Thomas Gorochowski, Johannes Roubos, René Verwaal

Research output: Contribution to journalArticle (Academic Journal)peer-review

11 Citations (Scopus)
376 Downloads (Pure)


High efficiency, ease of use and versatility of the CRISPR/Cas9 system has facilitated genetic modification of S. cerevisiae, a workhorse organism in biotechnology, extending its capability as a cell factory. Cas12a is an RNA-guided endonuclease with features distinguishable from Cas9, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single crRNA array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S. cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently coloured carotenoid producing yeast strains that were constructed.
Original languageEnglish
Article numbere59350
Number of pages15
JournalJournal of Visualized Experiments
Issue number147
Publication statusPublished - 28 May 2019

Structured keywords

  • Bristol BioDesign Institute
  • BrisSynBio


  • CRISPR/Cas12a
  • CRISPR/Cpf1
  • CRISPR/Cas9
  • multiplex genome editing
  • Saccharomyces cerevisiae
  • yeast pixel art


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