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

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
gene targeting
Yeast
Saccharomyces cerevisiae
Genes
synthetic genes
homologous recombination
cortisol
biochemical pathways
plasmids
Gene Knockout Techniques
genes
Nucleic Acid Repetitive Sequences
Homologous Recombination
Biosynthetic Pathways
RNA
Cortisol
yeasts
genome
Hydrocortisone

Keywords

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

ASJC Scopus subject areas

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

Cite this

Bao, Z., Xiao, H., Liang, J., Zhang, L., Xiong, X., Sun, N., ... 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.

Homology-integrated CRISPR-cas (HI-CRISPR) system for one-step multigene disruption in saccharomyces cerevisiae. / Bao, Zehua; Xiao, Han; Liang, Jing; Zhang, Lu; Xiong, Xiong; Sun, Ning; Si, Tong; Zhao, Huimin.

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

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

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. Food, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting, vol. 1, AIChE, pp. 27-36, Food, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting, Atlanta, United States, 11/16/14.
Bao Z, Xiao H, Liang J, Zhang L, Xiong X, Sun N et al. 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. AIChE. 2014. p. 27-36. (Food, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting).
Bao, Zehua ; Xiao, Han ; Liang, Jing ; Zhang, Lu ; Xiong, Xiong ; Sun, Ning ; Si, Tong ; Zhao, Huimin. / Homology-integrated CRISPR-cas (HI-CRISPR) system for one-step multigene disruption in saccharomyces cerevisiae. Food, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting. AIChE, 2014. pp. 27-36 (Food, Pharmaceutical and Bioengineering Division 2014 - Core Programming Area at the 2014 AIChE Annual Meeting).
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