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 Convenient Multiplexed Highly Efficient 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 Rapid Customized Genome Ediling 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 publicationFood, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting
PublisherAIChE
Pages27-36
Number of pages10
ISBN (Electronic)9781510812598
StatePublished - Jan 1 2014
EventFood, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting - Atlanta, United States
Duration: Nov 16 2014Nov 21 2014

Publication series

NameFood, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting
Volume1

Other

OtherFood, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area 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

  • Agricultural and Biological Sciences(all)
  • Pharmaceutical Science
  • Bioengineering

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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 Food, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting (pp. 27-36). (Food, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting; Vol. 1). AIChE.