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

Item Type:Article
Title:Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice
Creators Name:Spielmann, M. and Kakar, N. and Tayebi, N. and Leettola, C. and Nürnberg, G. and Sowada, N. and Lupiáñez, D.G. and Harabula, I. and Flöttmann, R. and Horn, D. and Chan, W.L. and Wittler, L. and Yilmaz, R. and Altmüller, J. and Thiele, H. and van Bokhoven, H. and Schwartz, C.E. and Nürnberg, P. and Bowie, J.U. and Ahmad, J. and Kubisch, C. and Mundlos, S. and Borck, G.
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.
Keywords:Amino Acid Sequence, Bacterial Proteins, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, Coculture Techniques, Congenital Limb Deformities, DNA Sequence Analysis, Endonucleases, Exome, Inbred C57BL Mice, Knockout Mice, Lod Score, MAP Kinase Kinase Kinases, Missense Mutation, Pedigree, Protein Kinases, Single Nucleotide Polymorphism, Site-Directed Mutagenesis, 129 Strain Mice, Animals, Mice
Source:Genome Research
Publisher:Cold Spring Harbor Laboratory Press
Page Range:183-191
Date:February 2016
Official Publication:https://doi.org/10.1101/gr.199430.115
PubMed:View item in PubMed

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