(664a) Enabling Multiplex Genome Engineering in Yeast By RNAi and Crispr

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.