Selection of chromosomal DNA libraries using a multiplex CRISPR system

  1. Owen W Ryan
  2. Jeffrey M Skerker
  3. Matthew J Maurer
  4. Xin Li
  5. Jordan C Tsai
  6. Snigdha Poddar
  7. Michael E Lee
  8. Will DeLoache
  9. John E Dueber
  10. Adam P Arkin
  11. Jamie H D Cate  Is a corresponding author
  1. BP Biofuels Global Technology Center, United States
  2. 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

  1. 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.
  2. Jeffrey M Skerker

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  3. Matthew J Maurer

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  4. Xin Li

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  5. Jordan C Tsai

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  6. Snigdha Poddar

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  7. Michael E Lee

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  8. Will DeLoache

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  9. John E Dueber

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  10. Adam P Arkin

    Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  11. 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.

Reviewing Editor

  1. Elisa Izaurralde, Max Planck Institute Development Biology, Germany

Publication history

  1. Received: June 16, 2014
  2. Accepted: August 17, 2014
  3. Accepted Manuscript published: August 19, 2014 (version 1)
  4. 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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  1. Owen W Ryan
  2. Jeffrey M Skerker
  3. Matthew J Maurer
  4. Xin Li
  5. Jordan C Tsai
  6. Snigdha Poddar
  7. Michael E Lee
  8. Will DeLoache
  9. John E Dueber
  10. Adam P Arkin
  11. Jamie H D Cate
(2014)
Selection of chromosomal DNA libraries using a multiplex CRISPR system
eLife 3:e03703.
https://doi.org/10.7554/eLife.03703

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