ACtivE: Assembly and CRISPR-targeted in vivo editing for yeast genome engineering using minimum reagents and time

Koray Malcı, Nestor Jonguitud-Borrego, Hugo van der Straten Waillet, Urtė Puodžiu Naitė, Emily J Johnston, Susan J Rosser, Leonardo Rios-Solis

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user's need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

Original languageEnglish
Pages (from-to)3629-3643
Number of pages15
JournalACS Synthetic Biology
Issue number11
Early online date17 Oct 2022
Publication statusPublished - 18 Nov 2022

Keywords / Materials (for Non-textual outputs)

  • saccharomyces cerevisiae
  • CRISPR toolkit
  • genome editing
  • synthetic biology
  • standardization
  • locus characterization


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