Effects of Cas9 expression on cell growth and production of natural products in Saccharomyces cerevisiae and optimization of CRISPR-Cas9 editing system

Abstract

CRISPR-Cas9 gene editing technology has been widely used in Saccharomyces cerevisiae.However, the effects of Cas9, as an exogenous protein, on the growth and production of natural products in S.cerevisiae are still unclear.In this study, Cas9 gene was expressed in S.cerevisiae by integration into the genome and construction into vectors, and two natural products, carotenoid and miltiradiene, were selected as the target products to study the effects of Cas9 expression on yeast growth and production capacity.The results showed that whether Cas9 was integrated into the genome or expressed by vectors, Cas9 inhibited the growth of S.cerevisiae, which was more obvious in the form of genome integration.When Cas9 was integrated into the genome, it had no effect on the production of carotenoid and miltiradiene by S.cerevisiae, but when Cas9 was expressed by vectors, the ability of S.cerevisiae to produce carotenoids and miltiradiene was significantly reduced.Therefore, in order to further efficiently knock out Cas9 after gene editing and minimize the adverse impact of Ura3 and Trp1 vectors, this study systematically explored the removal efficiency of the two vectors, and a plasmid capable of efficient gene editing was constructed, which optimized the application of CRISPR-Cas9 gene editing system in S.cerevisiae, and provided reference for the application of gene editing technology based on Cas9.