Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice

  1. Guntram Borck4
  1. 1Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany;
  2. 2Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, 13353 Berlin, Germany;
  3. 3Berlin-Brandenburg School for Regenerative Therapies (BSRT), 13353 Berlin, Germany;
  4. 4Institute of Human Genetics, University of Ulm, 89081 Ulm, Germany;
  5. 5International Graduate School in Molecular Medicine Ulm, University of Ulm, 89081 Ulm, Germany;
  6. 6Department of Biotechnology and Informatics, BUITEMS, Quetta, 57789 Pakistan;
  7. 7Department of Chemistry and Biochemistry, UCLA-DOE Institute of Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California 90095, USA;
  8. 8Cologne Center for Genomics, University of Cologne, 50931 Cologne, Germany;
  9. 9Berlin-Brandenburg Center for Regenerative Therapies (BCRT), 13353 Berlin, Germany;
  10. 10Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands;
  11. 11J.C. Self Research Institute, Greenwood Genetic Center, Greenwood, South Carolina 29646, USA;
  12. 12Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany;
  13. 13Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany;
  14. 14Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
  1. Corresponding authors: stefan.mundlos{at}charite.de, guntram.borck{at}uni-ulm.de

  1. ↵15 These authors contributed equally to this work.

Abstract

The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.

Footnotes

  • Received September 11, 2015.
  • Accepted December 7, 2015.

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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  1. Published in Advance January 11, 2016, doi: 10.1101/gr.199430.115 Genome Res. 26: 183-191 © 2016 Spielmann et al.; Published by Cold Spring Harbor Laboratory Press

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