CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in Saccharomyces cerevisiae

Abstract

Genomic integration is the preferred method for gene expression in microbial industrial production. However, traditional homologous recombination based multiplexed integration methods often suffer from low integration efficiency and complex experimental procedures. Here, we report a CRISPR/Cas9 based multiplexed integration (CMI) system in Saccharomyces cerevisiae, which can achieve quadruple integration at an individual locus without pre-engineering the host. A fused protein, Cas9-Brex27, was used as a bait to attract Rad51 recombinase to the proximity of the double-strand breaks introduced by the CRISPR/Cas9 system. The efficiency of quadruple integration was increased to 53.9% with 40 bp homology arms (HAs) and 78% with 100 bp HAs. CMI was applied to integrate a heterologous mogrol biosynthetic pathway consisting of four genes in a one-step transformation and offered an efficient solution for multiplexed integration. This method expands the synthetic biology toolbox of S. cerevisiae.

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools

“…35 Recently, the mogrol biosynthetic pathway has been integrated using the CMI system to generate an efficient producer within a week, significantly accelerating the process of metabolic engineering (Figure 4B). 67 Moreover, the gene drive delta site integration system by the CRISPR system (GDi-CRISPR) has been developed by combining the CRISPR system and gene drive principle (Figure 4B), which is another valuable tool for multicopy integration into the genome of S. cerevisiae. 152 Until now, the CRISPR system has been widely applied to tune metabolic processes such as the suppression of byproduct formation 153,154 and the introduction of pathways with multiple genes.…”

“…(A) Evolutionary relationship of the mutant strains and the metabolic strategies of S. cerevisiae in Yang et al’s study . (B) The principles of GDi-CRISPR and the CRISPR/Cas9 based multiplexed integration (CMI) systems in S. cerevisiae . (C) Utilizing CRISPR/dCas9-based CRISPRi or CRISPRa for metabolic regulation in a array or centralized form in S. cerevisiae .…”

“…As a result, a quadruple individual integration was obtained without pre-engineering the host of S. cerevisiae. 67 Multiplexing approaches can be efficiently used in polyploid S. cerevisiae as well but with reduced efficiency. For example, multiple gene knockouts were introduced in the polyploid strain ATCC 4124, resulting in 15−60% editing efficiency.…”

“…Based on the CMI method, local homologous recombination was enriched, and integration efficiency was promoted by attracting Rad51 proteins to DSB sites by the “bait” domain of Brex27. As a result, a quadruple individual integration was obtained without pre-engineering the host of S. cerevisiae …”

De Novo Biosynthesis of Free Vaccenic Acid with a Low Content of Oleic Acid in Saccharomyces cerevisiae

“…These results revealed that overexpression of CYB5 improves the activity of fatty acid desaturase CeFAT5, which is consistent with previous studies that a coexpression of CYB5 could increase the production of fatty acids by enhancing the desaturase activity. 48,49 Then, the CeFAT5 gene was integrated into the genomic site XI-2 50 of strain YJZ45H-GAL1, yielding strain YJZ45H-GAL1-FAT5. Subsequently, the plasmids pSH-ScCYB5 and pSH-ScCYB5-rELO1 were transformed into strain YJZ45H-GAL1-FAT5, yielding strains W13 and W14, respectively.…”