2. Academic Validation
  3. CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in Saccharomyces cerevisiae

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

  • ACS Synth Biol. 2023 Feb 28. doi: 10.1021/acssynbio.2c00591.
Jie Meng 1,  Yue Qiu 1,  Yueping Zhang 2,  Huimin Zhao 3,  Shuobo Shi 1
Affiliations

Affiliations

  • 1 Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
  • 2 College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
  • 3 Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
PMID: 36853024 DOI: 10.1021/acssynbio.2c00591
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.

Keywords

Brex27; CRISPR/Cas9; S. cerevisiae; metabolic engineering; multiplexed integration; synthetic biology.

Figures
Products