Selection of chromosomal DNA libraries using a multiplex CRISPR system

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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.

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Owen W Ryan

BP Biofuels Global Technology Center, San Diego, United States

Competing interests
Owen W Ryan, A patent application related to this work has been filed by J. Cate and O. Ryan on behalf of the Regents of the University of California.
Jeffrey M Skerker

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Matthew J Maurer

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Xin Li

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Jordan C Tsai

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Snigdha Poddar

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Michael E Lee

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Will DeLoache

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
John E Dueber

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Adam P Arkin

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Jamie H D Cate

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States

For correspondence
jcate@lbl.gov

Competing interests
Jamie H D Cate, A patent application related to this work has been filed by J. Cate and O. Ryan on behalf of the Regents of the University of California.

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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.