Development and expansion of the CRISPR/Cas9 toolboxes for powerful genome engineering in yeast.

Yeasts represent a group of the microorganisms most frequently seen in biotechnology. Recently, the class 2 type II CRISPR system (CRISPR/Cas9) has become the principal toolbox for genome editing. By efficiently implementing genetic manipulations such as gene integration/knockout, base editor, and transcription regulation, the development of biotechnological applications in yeasts has been extensively promoted. The genome-level tools based on CRISPR/Cas9, used for screening and identifying functional genes/gene clusters, are also advancing. In general, CRISPR/Cas9-assisted editing tools have gradually become standardized and function as host-orthogonal genetic systems, which results in time-saving for strain engineering and biotechnological application processes. In this review, we summarize the key points of the basic elements in the CRISPR/Cas9 system, including Cas9 variants, guide RNA, donors, and effectors. With a focus on yeast, we have also introduced the development of various CRISPR/Cas9 systems and discussed their future possibilities.