ACtivE: Assembly and CRISPR-targeted in vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time

ABSTRACT Among the numerous genetic tools developed for yeast, CRISPR/Cas system has been a widely used genome editing method thanks to its sophistication. However, CRISPR methods for yeast generally rely on pre-assembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose a convenient, rapid, standardizable CRISPR method, named Assembly and CRISPR-targeted in vivo Editing (ACtivE), which only relies on in vivo assembly of linear DNA fragments for both 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 ARS-close loci 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 β-carotene pathway. In only a few days, > 80% integration efficiency for single gene integration and > 50% integration efficiency for triplex integration were achieved 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.