(664a) Enabling Multiplex Genome Engineering in Yeast By RNAi and Crispr Conference: AIChE Annual MeetingYear: 2014Proceeding: 2014 AIChE Annual MeetingGroup: Food, Pharmaceutical & Bioengineering DivisionSession: Advances in Metabolic Engineering and Bioinformatics for Biofuels I: Strain Optimization Time: Thursday, November 20, 2014 - 12:30pm-12:48pm Authors: Si, T., University of Illinois at Urbana Champaign Zhao, H., University of Illinois at Urbana Champaign Lu, Z., University of Illinois at Champaign-Urbana Chao, R., University of Illinois at Champaign-Urbana Successful metabolic engineering practice often requires simultaneous manipulation of many genes. Whereas recombination-based genetic engineering (recombineering) is able to create combinatorial genetic diversity on a genome scale, such method is mainly limited to bacterial cells. Here we report a strategy that can generate multiplex modifications in the Saccharomyces cerevisiae genome, by combining the advances in full-length cDNA library construction, RNA-interference technology and CRISPR-Cas system. Briefly, upon introduction of specific double-stranded breaks (DSBs) in the repetitive sequences by CRISPR-Cas nucleases, both the overexpression and knockdown cassettes of every yeast gene can be integrated at high efficiency into the genomic loci of repetitive sequences. This process may be iteratively performed to accumulate dozens of genetic modifications in a single cell of an evolving yeast population. We fully automated this process through an integrated robotic platform, enabling generation of vast genetic diversity from which new or improved properties may emerge. We envision this new tool can greatly accelerate genome engineering in S. cerevisiae for basic and applied biological research and medicine.