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

Klaudia Ciurkot, Brenda Vonk, Thomas E. Gorochowski, J. Roubos, René Verwaal

Research output: Contribution to journalArticle

Abstract

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, 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 CRISPR RNA (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 colored carotenoid producing yeast strains that were constructed.

Original languageEnglish
JournalJournal of visualized experiments : JoVE
Issue number147
DOIs
Publication statusPublished - May 28 2019

Fingerprint

CRISPR-Associated Proteins
Clustered Regularly Interspaced Short Palindromic Repeats
Art
RNA
Yeast
Saccharomyces cerevisiae
Genes
Yeasts
Pixels
Proteins
DNA
DNA sequences
Genome
Guide RNA
Biotechnology
Gene Editing
Endonucleases
Design of experiments
Standardization
Carotenoids

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art. / Ciurkot, Klaudia; Vonk, Brenda; Gorochowski, Thomas E.; Roubos, J.; Verwaal, René.

In: Journal of visualized experiments : JoVE, No. 147, 28.05.2019.

Research output: Contribution to journalArticle

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