1. Developmental Biology
  2. Genetics and Genomics

Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction

  1. Alex Cornean
  2. Jakob Gierten
  3. Bettina Welz
  4. Juan Luis Mateo
  5. Thomas Thumberger
  6. Joachim Wittbrodt  Is a corresponding author
  1. Heidelberg University, Germany
  2. University of Oviedo, Spain
https://doi.org/10.7554/eLife.72124
Share this article
Cite this article
  1. Alex Cornean
  2. Jakob Gierten
  3. Bettina Welz
  4. Juan Luis Mateo
  5. Thomas Thumberger
  6. Joachim Wittbrodt
(2022)
Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction
eLife 11:e72124.
https://doi.org/10.7554/eLife.72124
  2. Download BibTeX
  3. Download .RIS

Abstract

Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.

Data availability

Source code for ACEofBASEs has been provided.

Article and author information

Author details

  1. Alex Cornean

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3727-7057

  2. Jakob Gierten

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.

  3. Bettina Welz

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.

  4. Juan Luis Mateo

    Deparment of Computer Science, University of Oviedo, Oviedo, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9902-6048

  5. Thomas Thumberger

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8485-457X

  6. Joachim Wittbrodt

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    For correspondence
    jochen.wittbrodt@cos.uni-heidelberg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8550-7377

Funding

Deutsche Forschungsgemeinschaft (WI 1824/9-1)

  • Joachim Wittbrodt

H2020 European Research Council (810172)

  • Joachim Wittbrodt

Deutsches Zentrum für Herz-Kreislaufforschung

  • Joachim Wittbrodt

Deutsche Herzstiftung (S/02/17)

  • Jakob Gierten

Joachim Herz Stiftung

  • Jakob Gierten

Deutsche Forschungsgemeinschaft (3DMM2O)

  • Joachim Wittbrodt

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

Reviewing Editor

  1. Stephen C Ekker, Mayo Clinic, United States

Version history

  1. Received: July 12, 2021
  2. Preprint posted: July 26, 2021 (view preprint)
  3. Accepted: March 21, 2022
  4. Accepted Manuscript published: April 4, 2022 (version 1)
  5. Version of Record published: April 22, 2022 (version 2)

Copyright

© 2022, Cornean 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

  • 2,568
    Page views
  • 296
    Downloads
  • 8
    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)

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. Alex Cornean
  2. Jakob Gierten
  3. Bettina Welz
  4. Juan Luis Mateo
  5. Thomas Thumberger
  6. Joachim Wittbrodt
(2022)
Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction
eLife 11:e72124.
https://doi.org/10.7554/eLife.72124

Categories and tags

Research organism

Further reading

    1. Developmental Biology

    Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch in C. elegans

    Taylor N Medwig-Kinney, Brian A Kinney ... David Q Matus
    Research Article

    A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

    1. Chromosomes and Gene Expression
    2. Developmental Biology

    Transcription: A hub of activity

    Virginia L Pimmett, Mounia Lagha
    Insight

    Imaging experiments reveal the complex and dynamic nature of the transcriptional hubs associated with Notch signaling.

    1. Cell Biology
    2. Developmental Biology

    Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human

    Simon Schneider, Andjela Kovacevic ... Hubert Schorle
    Research Article

    Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.