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

Genome Res. 2016 Feb;26(2):183-91. doi: 10.1101/gr.199430.115.

Malte Spielmann ¹, Naseebullah Kakar ², Naeimeh Tayebi ³, Catherine Leettola ⁴, Gudrun Nürnberg ⁵, Nadine Sowada ⁶, Darío G Lupiáñez ⁷, Izabela Harabula ³, Ricarda Flöttmann ⁸, Denise Horn ⁸, Wing Lee Chan ⁸, Lars Wittler ³, Rüstem Yilmaz ⁶, Janine Altmüller ⁵, Holger Thiele ⁵, Hans van Bokhoven ⁹, Charles E Schwartz ¹⁰, Peter Nürnberg ¹¹, James U Bowie ⁴, Jamil Ahmad ¹², Christian Kubisch ¹³, Stefan Mundlos ¹, Guntram Borck ¹⁴

Affiliations

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

PMID: 26755636 DOI: 10.1101/gr.199430.115

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.

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