TY - JOUR TI - Selection of chromosomal DNA libraries using a multiplex CRISPR system AU - Ryan, Owen W AU - Skerker, Jeffrey M AU - Maurer, Matthew J AU - Li, Xin AU - Tsai, Jordan C AU - Poddar, Snigdha AU - Lee, Michael E AU - DeLoache, Will AU - Dueber, John E AU - Arkin, Adam P AU - Cate, Jamie HD A2 - Izaurralde, Elisa VL - 3 PY - 2014 DA - 2014/08/19 SP - e03703 C1 - eLife 2014;3:e03703 DO - 10.7554/eLife.03703 UR - https://doi.org/10.7554/eLife.03703 AB - The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function. KW - directed evolution KW - chromosome KW - CRISPR KW - Cas9 KW - genome editing JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -