Abstract

CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.

Data availability

All data generated are available in Supplementary file 2.

Article and author information

Author details

  1. Jackson Champer

    Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States
    For correspondence
    jc3248@cornell.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3814-3774
  2. Joan Chung

    Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Yoo Lim Lee

    Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Chen Liu

    Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Emily Yang

    Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Zhaoxin Wen

    Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Andrew G Clark

    Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Philipp W Messer

    Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States
    For correspondence
    messer@cornell.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8453-9377

Funding

National Institutes of Health (R21AI130635)

  • Jackson Champer
  • Andrew G Clark
  • Philipp W Messer

National Institutes of Health (F32AI138476)

  • Jackson Champer

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

Copyright

© 2019, Champer 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

  • 5,222
    views
  • 667
    downloads
  • 107
    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. Jackson Champer
  2. Joan Chung
  3. Yoo Lim Lee
  4. Chen Liu
  5. Emily Yang
  6. Zhaoxin Wen
  7. Andrew G Clark
  8. Philipp W Messer
(2019)
Molecular safeguarding of CRISPR gene drive experiments
eLife 8:e41439.
https://doi.org/10.7554/eLife.41439

Share this article

https://doi.org/10.7554/eLife.41439

Further reading

    1. Genetics and Genomics
    2. Immunology and Inflammation
    Stephanie Guillet, Tomi Lazarov ... Frédéric Geissmann
    Research Article

    Systemic lupus erythematosus (SLE) is an autoimmune disease, the pathophysiology and genetic basis of which are incompletely understood. Using a forward genetic screen in multiplex families with SLE, we identified an association between SLE and compound heterozygous deleterious variants in the non-receptor tyrosine kinases (NRTKs) ACK1 and BRK. Experimental blockade of ACK1 or BRK increased circulating autoantibodies in vivo in mice and exacerbated glomerular IgG deposits in an SLE mouse model. Mechanistically, NRTKs regulate activation, migration, and proliferation of immune cells. We found that the patients’ ACK1 and BRK variants impair efferocytosis, the MERTK-mediated anti-inflammatory response to apoptotic cells, in human induced pluripotent stem cell (hiPSC)-derived macrophages, which may contribute to SLE pathogenesis. Overall, our data suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis in macrophages.

    1. Genetics and Genomics
    2. Microbiology and Infectious Disease
    Dániel Molnár, Éva Viola Surányi ... Judit Toth
    Research Article

    The sustained success of Mycobacterium tuberculosis as a pathogen arises from its ability to persist within macrophages for extended periods and its limited responsiveness to antibiotics. Furthermore, the high incidence of resistance to the few available antituberculosis drugs is a significant concern, especially since the driving forces of the emergence of drug resistance are not clear. Drug-resistant strains of Mycobacterium tuberculosis can emerge through de novo mutations, however, mycobacterial mutation rates are low. To unravel the effects of antibiotic pressure on genome stability, we determined the genetic variability, phenotypic tolerance, DNA repair system activation, and dNTP pool upon treatment with current antibiotics using Mycobacterium smegmatis. Whole-genome sequencing revealed no significant increase in mutation rates after prolonged exposure to first-line antibiotics. However, the phenotypic fluctuation assay indicated rapid adaptation to antibiotics mediated by non-genetic factors. The upregulation of DNA repair genes, measured using qPCR, suggests that genomic integrity may be maintained through the activation of specific DNA repair pathways. Our results, indicating that antibiotic exposure does not result in de novo adaptive mutagenesis under laboratory conditions, do not lend support to the model suggesting antibiotic resistance development through drug pressure-induced microevolution.