Structural variations (SVs) contribute to the variability of our genome and are often associated with disease. Their study in model systems was hampered until now by labor-intensive genetic targeting procedures and multiple mouse crossing steps. Here we present the use of CRISPR/Cas for the fast (10 weeks) and efficient generation of SVs in mice. We specifically produced deletions, inversions, and also duplications at six different genomic loci ranging from 1.1 kb to 1.6 Mb with efficiencies up to 42%. After PCR-based selection, clones were successfully used to create mice via aggregation. To test the practicability of the method, we reproduced a human 500 kb disease-associated deletion and were able to recapitulate the human phenotype in mice. Furthermore, we evaluated the regulatory potential of a large genomic interval by deleting a 1.5 Mb fragment. The method presented permits rapid in vivo modeling of genomic rearrangements.
Copy number variations (CNVs) often include non-coding sequence and putative enhancers but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of Indian hedgehog (IHH), causing multiple, highly localised phenotypes including craniosynostosis and synpolydactyly1,2. We show through transgenic reporter and genome editing studies in mice that Ihh is regulated by a constellation of at least 9 enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions show that they function in an additive manner resulting in growth defects of the skull and long bones. Duplications, in contrast, cause not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatio-temporal control of developmental gene expression is achieved by complex multipartite enhancer ensembles. Alterations in the composition of such clusters can result in gene misexpression and disease.
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