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

Version 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.

Metrics

  • 13,464
    Page views
  • 2,460
    Downloads
  • 265
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Share this article

https://doi.org/10.7554/eLife.03703

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Karolina Honzejkova, Dalibor Kosek ... Tomas Obsil
    Research Article

    Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Daniyal Tariq, Nicole Maurici ... Brian R Crane
    Research Article

    Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid–liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.