Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a

Klaudia W Ciurkot, Thomas E Gorochowski, Johannes A Roubos, René Verwaal

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

Abstract

CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.
Original languageEnglish
Article numbergkab529
Number of pages16
JournalNucleic Acids Research
Volume49
Issue number13
Early online date1 Jul 2021
DOIs
Publication statusPublished - 21 Jul 2021

Structured keywords

  • BrisSynBio
  • Bristol BioDesign Institute

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