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.