title: Selection of chromosomal DNA libraries using a multiplex CRISPR system.
abstract: 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.
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Owen W
authors.initials: OW
authors.lastname: Ryan
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Skerker
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Maurer
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Jordan C
authors.initials: JC
authors.lastname: Tsai
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Snigdha
authors.initials: S
authors.lastname: Poddar
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Lee
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Will
authors.initials: W
authors.lastname: DeLoache
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: John E
authors.initials: JE
authors.lastname: Dueber
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.
authors.forename: Jamie H D
authors.initials: JH
authors.lastname: Cate
mesh_terms: D000483:Alleles; D002475:Cellobiose / Q000737:chemistry; D002875:Chromosomes / Q000648:ultrastructure*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005285:Fermentation; D015723:Gene Library; D005818:Genetic Engineering; D005821:Genetic Techniques; D016681:Genome, Fungal; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D002475:Cellobiose
keywords: CRISPR; Cas9; S. cerevisiae; biophysics; chromosome; chromosomes; directed evolution; genes; genome editing; structural biology