title: Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.
abstract: One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.
authors.affiliation: †Department of Biochemistry, ‡Department of Chemical and Biomolecular Engineering, §Departments of Chemistry, and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Zehua
authors.initials: Z
authors.lastname: Bao
authors.affiliation: †Department of Biochemistry, ‡Department of Chemical and Biomolecular Engineering, §Departments of Chemistry, and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Han
authors.initials: H
authors.lastname: Xiao
authors.affiliation: †Department of Biochemistry, ‡Department of Chemical and Biomolecular Engineering, §Departments of Chemistry, and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Liang
authors.affiliation: †Department of Biochemistry, ‡Department of Chemical and Biomolecular Engineering, §Departments of Chemistry, and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: †Department of Biochemistry, ‡Department of Chemical and Biomolecular Engineering, §Departments of Chemistry, and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Xiong
authors.initials: X
authors.lastname: Xiong
authors.affiliation: †Department of Biochemistry, ‡Department of Chemical and Biomolecular Engineering, §Departments of Chemistry, and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Ning
authors.initials: N
authors.lastname: Sun
authors.affiliation: †Department of Biochemistry, ‡Department of Chemical and Biomolecular Engineering, §Departments of Chemistry, and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Tong
authors.initials: T
authors.lastname: Si
authors.affiliation: †Department of Biochemistry, ‡Department of Chemical and Biomolecular Engineering, §Departments of Chemistry, and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000235:genetics; D001483:Base Sequence; D053898:Biosynthetic Pathways / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D059765:Homologous Recombination / Q000235:genetics*; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D026923:Amino Acid Transport Systems, Basic; C502212:CAN1 protein, S cerevisiae; C430023:LYP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA
keywords: CRISPR−Cas; Saccharomyces cerevisiae; gene knockout; genome editing; multiple gene disruption