Homology-integrated CRISPR-cas (HI-CRISPR) system for one-step multigene disruption in saccharomyces cerevisiae

Zehua Bao, Han Xiao, Jing Liang, Lu Zhang, Xiong Xiong, Ning Sun, Tong Si, Huimin Zhao

Research output: Chapter in Book/Report/Conference proceedingConference contribution

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.

Original languageEnglish (US)
Title of host publicationSystems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting
PublisherAIChE
Pages3-12
Number of pages10
ISBN (Electronic)9781510812475
StatePublished - Jan 1 2014
EventSystems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting - Atlanta, United States
Duration: Nov 16 2014Nov 21 2014

Publication series

NameSystems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting

Other

OtherSystems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting
CountryUnited States
CityAtlanta
Period11/16/1411/21/14

Keywords

  • CRISPR-Cas
  • Gene knockout
  • Genome editing
  • Multiple gene disruption
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Renewable Energy, Sustainability and the Environment
  • Molecular Biology
  • Structural Biology
  • Biomedical Engineering

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  • Cite this

    Bao, Z., Xiao, H., Liang, J., Zhang, L., Xiong, X., Sun, N., Si, T., & Zhao, H. (2014). Homology-integrated CRISPR-cas (HI-CRISPR) system for one-step multigene disruption in saccharomyces cerevisiae. In Systems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting (pp. 3-12). (Systems Biology 2014 - Topical Conference at the 2014 AIChE Annual Meeting). AIChE.