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ACS Synth Biol. 2015 May 15;4(5):585-94. doi: 10.1021/sb500255k. Epub 2014 Sep 19.

Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

Author information

1
†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.

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.

KEYWORDS:

CRISPR−Cas; Saccharomyces cerevisiae; gene knockout; genome editing; multiple gene disruption

PMID:
25207793
DOI:
10.1021/sb500255k
[Indexed for MEDLINE]

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