1. Immunology and Inflammation
  2. Microbiology and Infectious Disease
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Reprogramming the antigen specificity of B cells using genome-editing technologies

  1. James E Voss  Is a corresponding author
  2. Alicia Gonzalez-Martin  Is a corresponding author
  3. Raiees Andrabi
  4. Roberta P Fuller
  5. Ben Murrell
  6. Laura E McCoy
  7. Katelyn Porter
  8. Deli Huang
  9. Wenjuan Li
  10. Devin Sok
  11. Khoa Le
  12. Bryan Briney
  13. Morgan Chateau
  14. Geoffrey Rogers
  15. Lars Hangartner
  16. Ann J Feeney
  17. David Nemazee
  18. Paula Cannon
  19. Dennis Burton  Is a corresponding author
  1. The Scripps Research Institute, United States
  2. Universidad Autónoma de Madrid (UAM), Spain
  3. Karolinska Institutet, Sweden
  4. University College London, United Kingdom
  5. University of Southern California, United States
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Cite this article as: eLife 2019;8:e42995 doi: 10.7554/eLife.42995
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Abstract

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope by FACS and sequenced mRNA from these cells showed PG9 HC expressed as several different isotypes after culture with CD40 ligand and IL-4.

Data availability

Next generation sequencing data from RT-PCR amplicons have been deposited at Dryad:DOI: https://doi.org/10.5061/dryad.45j0r70.Amplification free whole genome sequencing reads mapped to the human reference genome have been deposited to NCBI with BioSample accession numbers SAMN09404498 and SAMN09404497

The following data sets were generated

Article and author information

Author details

  1. James E Voss

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    For correspondence
    jvoss@scripps.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4777-1596
  2. Alicia Gonzalez-Martin

    Department of Biochemistry, Universidad Autónoma de Madrid (UAM), Madrid, Spain
    For correspondence
    alicia.gonzalezm@uam.es
    Competing interests
    The authors declare that no competing interests exist.
  3. Raiees Andrabi

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Roberta P Fuller

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ben Murrell

    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
    Competing interests
    The authors declare that no competing interests exist.
  6. Laura E McCoy

    Division of Infection and Immunity, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Katelyn Porter

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Deli Huang

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Wenjuan Li

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Devin Sok

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Khoa Le

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Bryan Briney

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Morgan Chateau

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Geoffrey Rogers

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Lars Hangartner

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. Ann J Feeney

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. David Nemazee

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Paula Cannon

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  19. Dennis Burton

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    For correspondence
    burton@scripps.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institutes of Health (5R01DE025167-05)

  • Dennis Burton

Bill and Melinda Gates Foundation (OPP1183956)

  • James E Voss

Ramón y Cajal Merit Award, Ministerio de Ciencia, Innovacion y Universidades (RYC-2016-21155)

  • Alicia Gonzalez-Martin

Marie-Curie Fellowship (FP7-PEOPLE-2013-IOF)

  • Laura E McCoy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Tomohiro Kurosaki, Osaka University, Japan

Publication history

  1. Received: October 19, 2018
  2. Accepted: December 31, 2018
  3. Accepted Manuscript published: January 16, 2019 (version 1)
  4. Accepted Manuscript updated: January 17, 2019 (version 2)
  5. Version of Record published: January 31, 2019 (version 3)

Copyright

© 2019, Voss 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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