CRISPR/Cas9‐RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high‐performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double‐strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis‐muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.

Development and Application of CRISPR/Cas in Microbial Biotechnology

“…By using this method, 15 exogenous DNA parts were correctly assembled and integrated into 3 genomic loci for carotenoid production in one transformation ( Jakociunas et al, 2015 , 2018a ). Kildegaard adopted similar strategy for multi-architecture assembly and insertion ( Kildegaard et al, 2019 ). Kuivanen et al (2018) reported a high-throughput workflow for CRISPR/Cas9 mediated combinatorial promoter replacements, and successfully edited 3 loci simultaneously with a frequency of 50%.…”

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

“…Subsequently, a number of studies have employed CRISPR/Cas9 strategy to study genes that may contribute to fungal virulence [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Interestingly, Kildergaard et al, combined the CRISPR/Cas9 system with RNA-interference (RNA-i) to generate simultaneous mutations in multiple genes [ 20 ]. Similarly, CRISPR/Cas9 systems have been employed to generate multiple simultaneous mutations with high efficiencies [ 21 , 22 , 23 , 24 ].…”

Advances in RNAi-Assisted Strain Engineering in Saccharomyces cerevisiae

“…Heterologous RNAi pathways from S. castelli and human were successfully reconstituted in S. cerevisiae . To evaluate RNAi effectiveness and efficiency, fluorescent proteins were commonly utilized as reporters ( Drinnenberg et al, 2009 ; Suk et al, 2011 ; Crook et al, 2014 ; Si et al, 2014 ; Purcell et al, 2018 ; Kildegaard et al, 2019 ). For the S. castelli pathway, robust repression of green fluorescent protein (GFP) signals were observed when both DCR1 and AGO1 were present, but DCR1 alone was sufficient to generate GFP siRNAs in S. cerevisiae ( Drinnenberg et al, 2009 ).…”