1. Structural Biology and Molecular Biophysics
  2. Chromosomes and Gene Expression

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
https://doi.org/10.7554/eLife.03703
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

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

  • 13,597
    views
  • 2,583
    downloads
  • 319
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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

Categories and tags

Research organism

Further reading

    1. Structural Biology and Molecular Biophysics

    Functionally important residues from graph analysis of coevolved dynamic couplings

    Manming Xu, Sarath Chandra Dantu ... Shozeb Haider
    Research Article

    The relationship between protein dynamics and function is essential for understanding biological processes and developing effective therapeutics. Functional sites within proteins are critical for activities such as substrate binding, catalysis, and structural changes. Existing computational methods for the predictions of functional residues are trained on sequence, structural, and experimental data, but they do not explicitly model the influence of evolution on protein dynamics. This overlooked contribution is essential as it is known that evolution can fine-tune protein dynamics through compensatory mutations either to improve the proteins’ performance or diversify its function while maintaining the same structural scaffold. To model this critical contribution, we introduce DyNoPy, a computational method that combines residue coevolution analysis with molecular dynamics simulations, revealing hidden correlations between functional sites. DyNoPy constructs a graph model of residue–residue interactions, identifies communities of key residue groups, and annotates critical sites based on their roles. By leveraging the concept of coevolved dynamical couplings—residue pairs with critical dynamical interactions that have been preserved during evolution—DyNoPy offers a powerful method for predicting and analysing protein evolution and dynamics. We demonstrate the effectiveness of DyNoPy on SHV-1 and PDC-3, chromosomally encoded β-lactamases linked to antibiotic resistance, highlighting its potential to inform drug design and address pressing healthcare challenges.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    AI-based discovery and cryoEM structural elucidation of a KATP channel pharmacochaperone

    Assmaa Elsheikh, Camden M Driggers ... Show-Ling Shyng
    Research Article

    Pancreatic KATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the KATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used KATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for KATP trafficking-impaired CHI is hindered by high affinity binding, which limits functional recovery of rescued channels. Recent structural studies of KATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known KATP pharmacochaperones bind. The structural knowledge provides a framework for discovering KATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on KATP channel trafficking mutations. Aekatperone reversibly inhibits KATP channel activity with a half-maximal inhibitory concentration (IC50) ~9 μM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of KATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a KATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by KATP trafficking defects.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Microphase separation produces interfacial environment within diblock biomolecular condensates

    Andrew P Latham, Longchen Zhu ... Bin Zhang
    Research Article

    The phase separation of intrinsically disordered proteins is emerging as an important mechanism for cellular organization. However, efforts to connect protein sequences to the physical properties of condensates, that is, the molecular grammar, are hampered by a lack of effective approaches for probing high-resolution structural details. Using a combination of multiscale simulations and fluorescence lifetime imaging microscopy experiments, we systematically explored a series of systems consisting of diblock elastin-like polypeptides (ELPs). The simulations succeeded in reproducing the variation of condensate stability upon amino acid substitution and revealed different microenvironments within a single condensate, which we verified with environmentally sensitive fluorophores. The interspersion of hydrophilic and hydrophobic residues and a lack of secondary structure formation result in an interfacial environment, which explains both the strong correlation between ELP condensate stability and interfacial hydrophobicity scales, as well as the prevalence of protein-water hydrogen bonds. Our study uncovers new mechanisms for condensate stability and organization that may be broadly applicable.