Genome editing systems across yeast species

Abstract

Yeasts are used to produce a myriad of value-added compounds. Engineering yeasts into cost-efficient cell factories is greatly facilitated by the availability of genome editing tools. While traditional engineering techniques such as homologous recombination-based gene knockout and pathway integration continue to be widely used, novel genome editing systems including multiplexed approaches, bacteriophage integrases, CRISPR-Cas systems, and base editors are emerging as more powerful toolsets to accomplish rapid genome scale engineering and phenotype screening. In this review, we summarized the techniques which have been successfully implemented in model yeast Saccharomyces cerevisiae as well as non-conventional yeast species. The mechanisms and applications of various genome engineering systems are discussed and general guidelines to expand genome editing systems from S. cerevisiae to other yeast species are also highlighted.

Figure 1.. Schematic representation of yeast genome editing techniques.

(A) HR and Cre-loxP system. Integration cassette with selection marker flanked by loxP sites is integrated into genome via HR. Selection marker is cured by expression of Cre recombinase. (B) Serine integrase mediated marker less integration. Plasmid containing serine integrase and integration cassette flanked by attP sites of serine integrase is transformed into yeast installed with attB sites of serine integrase. Expression of serine integrase could mediate recombination between attB and attP sites to integrase the cassette. (C) TALEN mediated genome engineering in yeast. TALEN consists of a DNA binding domain and a FokI nuclease domain. DSB is repaired by NHEJ to result in indel mutations or HR to achieve knock-in. (D) Multiplexed genome editing using CRISPR-Cas. Delivery of Cas effector and gRNA array can be achieved on a single plasmid. gRNA array is separated by a spacer such as tRNA to facilitate the gRNA processing. Simultaneous gene knockout and cassette knock-in is achieved.