Selection of chromosomal DNA libraries using a multiplex CRISPR system
- BP Biofuels Global Technology Center, United States
- Energy Biosciences Institute, University of California, Berkeley, United States
Abstract
The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over ten-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.
Article and author information
Author details
Reviewing Editor
- Elisa Izaurralde, Max Planck Institute Development Biology, Germany
Version history
- Received: June 16, 2014
- Accepted: August 17, 2014
- Accepted Manuscript published: August 19, 2014 (version 1)
- Version of Record published: September 15, 2014 (version 2)
Copyright
© 2014, Ryan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Selection of chromosomal DNA libraries using a multiplex CRISPR system
eLife 3:e03703.
https://doi.org/10.7554/eLife.03703
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