title: Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.
abstract: Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.|Muse Biotechnology Inc., Boulder, Colorado, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Garst
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.|Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado, USA.
authors.forename: Marcelo C
authors.initials: MC
authors.lastname: Bassalo
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
authors.forename: Gur
authors.initials: G
authors.lastname: Pines
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
authors.forename: Sean A
authors.initials: SA
authors.lastname: Lynch
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.|Muse Biotechnology Inc., Boulder, Colorado, USA.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Halweg-Edwards
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
authors.forename: Rongming
authors.initials: R
authors.lastname: Liu
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
authors.forename: Liya
authors.initials: L
authors.lastname: Liang
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.|Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, People's Republic of China.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, People's Republic of China.|Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, People's Republic of China.
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
authors.forename: Ramsey
authors.initials: R
authors.lastname: Zeitoun
authors.affiliation: Muse Biotechnology Inc., Boulder, Colorado, USA.
authors.forename: William G
authors.initials: WG
authors.lastname: Alexander
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
mesh_terms: D000465:Algorithms; D002874:Chromosome Mapping / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D000072669:Gene Editing / Q000379:methods*; D016680:Genome, Bacterial / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D060847:Metabolic Engineering / Q000379:methods*; D055442:Metabolome / Q000235:genetics; D009711:Nucleotides / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D020543:Proteome / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012984:Software
chemical_list: D009711:Nucleotides; D020543:Proteome