Reprogramming the antigen specificity of B cells using genome-editing technologies
- The Scripps Research Institute, United States
- Universidad Autónoma de Madrid (UAM), Spain
- Karolinska Institutet, United States
- University College London, United Kingdom
- University of Southern California, United States
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
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PG9HC(V434)Ramos-WGSNCBI SRA, SAMN09404498.
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PG9HC(V781)Ramos-WGSNCBI SRA, SAMN09404497.
Article and author information
Author details
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
- Tomohiro Kurosaki, Osaka University, Japan
Version history
- Received: October 19, 2018
- Accepted: December 31, 2018
- Accepted Manuscript published: January 16, 2019 (version 1)
- Accepted Manuscript updated: January 17, 2019 (version 2)
- 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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Reprogramming the antigen specificity of B cells using genome-editing technologies
eLife 8:e42995.
https://doi.org/10.7554/eLife.42995
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