1. Immunology and Inflammation
  2. Microbiology and Infectious Disease
Download icon

CRISPR-based functional genomics in human dendritic cells

  1. Marco Jost  Is a corresponding author
  2. Amy N Jacobson
  3. Jeffrey A Hussmann
  4. Giana Cirolia
  5. Michael A Fischbach  Is a corresponding author
  6. Jonathan S Weissman  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Stanford University, United States
  3. Chan Zuckerberg Biohub, United States
Tools and Resources
  • Cited 0
  • Views 2,143
  • Annotations
Cite this article as: eLife 2021;10:e65856 doi: 10.7554/eLife.65856

Abstract

Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.

Data availability

Raw data from RNA-seq of unedited and edited moDCs are available at GEO under accession codes GSE161401 and GSE161466, respectively. Raw data from amplicon sequencing for all samples are available at SRA under accession code PRJNA673198. Processed data from amplicon sequencing as well as raw and processed data from the genetic screens are provided as supplemental files (Supplementary Files 2, 3, 4).

The following data sets were generated

Article and author information

Author details

  1. Marco Jost

    Cellular and Molecular Pharmacology / Microbiology and Immunology, University of California, San Francisco, San Francisco, United States
    For correspondence
    marco.jost@gmail.com
    Competing interests
    Marco Jost, MJ consults for Maze Therapeutics..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1369-4908
  2. Amy N Jacobson

    Department of Bioengineering / ChEM-H, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  3. Jeffrey A Hussmann

    Cellular and Molecular Pharmacology / Microbiology and Immunology, University of California, San Francisco, San Francisco, United States
    Competing interests
    Jeffrey A Hussmann, JAH consults for Tessera Therapeutics..
  4. Giana Cirolia

    NA, Chan Zuckerberg Biohub, San Francisco, United States
    Competing interests
    No competing interests declared.
  5. Michael A Fischbach

    Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States
    For correspondence
    fischbach@fischbachgroup.org
    Competing interests
    Michael A Fischbach, MAF is a co-founder and director of Federation Bio and Viralogic..
  6. Jonathan S Weissman

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    For correspondence
    Jonathan.Weissman@ucsf.edu
    Competing interests
    Jonathan S Weissman, JSW consults for and holds equity in KSQ Therapeutics, Maze Therapeutics, and Tenaya Therapeutics. JSW is a venture partner at 5AM Ventures and a member of the Amgen Scientific Advisory Board..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2445-670X

Funding

National Institutes of Health (K99 GM130964)

  • Marco Jost

Damon Runyon Cancer Research Foundation (DRG-2262-16)

  • Jeffrey A Hussmann

Howard Hughes Medical Institute (Investigator)

  • Jonathan S Weissman

National Institutes of Health (U01 CA217882)

  • Jonathan S Weissman

National Institutes of Health (DP1 DK113598)

  • Michael A Fischbach

National Institutes of Health (R01 DK11017404)

  • Michael A Fischbach

Chan-Zuckerberg Initiative

  • Jonathan S Weissman

Helmsley Foundation

  • Michael A Fischbach

Howard Hughes Medical Institute (Simons Faculty Scholar Award)

  • Michael A Fischbach

Burroughs Wellcome Fund (Investigators in the Pathogenesis of Infectious Disease program)

  • Michael A Fischbach

UCSF Program for Breakthrough Biomedical Research (Postdoctoral Independent Research Grant)

  • Marco Jost

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

Reviewing Editor

  1. Florent Ginhoux, Agency for Science Technology and Research, Singapore

Publication history

  1. Received: December 16, 2020
  2. Accepted: April 26, 2021
  3. Accepted Manuscript published: April 27, 2021 (version 1)
  4. Version of Record published: May 7, 2021 (version 2)

Copyright

© 2021, Jost 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

  • 2,143
    Page views
  • 341
    Downloads
  • 0
    Citations

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

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)

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

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

Further reading

    1. Cell Biology
    2. Immunology and Inflammation
    Janine JG Arts et al.
    Research Article Updated

    Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

    1. Epidemiology and Global Health
    2. Immunology and Inflammation
    Christina M Bergey
    Insight

    The immune cells of macaques fed a Western-like diet adopt a pro-inflammatory profile.