title: Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice.
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.
authors.affiliation: 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;
authors.forename: Malte
authors.initials: M
authors.lastname: Spielmann
authors.affiliation: 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;
authors.forename: Naseebullah
authors.initials: N
authors.lastname: Kakar
authors.affiliation: Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany;
authors.forename: Naeimeh
authors.initials: N
authors.lastname: Tayebi
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute of Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California 90095, USA;
authors.forename: Catherine
authors.initials: C
authors.lastname: Leettola
authors.affiliation: Cologne Center for Genomics, University of Cologne, 50931 Cologne, Germany;
authors.forename: Gudrun
authors.initials: G
authors.lastname: Nürnberg
authors.affiliation: Institute of Human Genetics, University of Ulm, 89081 Ulm, Germany; International Graduate School in Molecular Medicine Ulm, University of Ulm, 89081 Ulm, Germany;
authors.forename: Nadine
authors.initials: N
authors.lastname: Sowada
authors.affiliation: 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;
authors.forename: Darío G
authors.initials: DG
authors.lastname: Lupiáñez
authors.affiliation: Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany;
authors.forename: Izabela
authors.initials: I
authors.lastname: Harabula
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, 13353 Berlin, Germany;
authors.forename: Ricarda
authors.initials: R
authors.lastname: Flöttmann
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, 13353 Berlin, Germany;
authors.forename: Denise
authors.initials: D
authors.lastname: Horn
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, 13353 Berlin, Germany;
authors.forename: Wing Lee
authors.initials: WL
authors.lastname: Chan
authors.affiliation: Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany;
authors.forename: Lars
authors.initials: L
authors.lastname: Wittler
authors.affiliation: Institute of Human Genetics, University of Ulm, 89081 Ulm, Germany; International Graduate School in Molecular Medicine Ulm, University of Ulm, 89081 Ulm, Germany;
authors.forename: Rüstem
authors.initials: R
authors.lastname: Yilmaz
authors.affiliation: Cologne Center for Genomics, University of Cologne, 50931 Cologne, Germany;
authors.forename: Janine
authors.initials: J
authors.lastname: Altmüller
authors.affiliation: Cologne Center for Genomics, University of Cologne, 50931 Cologne, Germany;
authors.forename: Holger
authors.initials: H
authors.lastname: Thiele
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands;
authors.forename: Hans
authors.initials: H
authors.lastname: van Bokhoven
authors.affiliation: J.C. Self Research Institute, Greenwood Genetic Center, Greenwood, South Carolina 29646, USA;
authors.forename: Charles E
authors.initials: CE
authors.lastname: Schwartz
authors.affiliation: 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;
authors.forename: Peter
authors.initials: P
authors.lastname: Nürnberg
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute of Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California 90095, USA;
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
authors.affiliation: Department of Biotechnology and Informatics, BUITEMS, Quetta, 57789 Pakistan;
authors.forename: Jamil
authors.initials: J
authors.lastname: Ahmad
authors.affiliation: Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Kubisch
authors.affiliation: 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;
authors.forename: Stefan
authors.initials: S
authors.lastname: Mundlos
authors.affiliation: Institute of Human Genetics, University of Ulm, 89081 Ulm, Germany;
authors.forename: Guntram
authors.initials: G
authors.lastname: Borck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018920:Coculture Techniques; D004720:Endonucleases; D059472:Exome; D005260:Female; D006801:Humans; D017880:Limb Deformities, Congenital / Q000235:genetics*; D008126:Lod Score; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry / Q000235:genetics*; D008297:Male; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; D004720:Endonucleases; D020930:MAP Kinase Kinase Kinases; D011494:Protein Kinases; C413047:MAP3K20 protein, human; C581706:Map3k20 protein, mouse; C000606107:Cas9 endonuclease Streptococcus pyogenes