Pathogenic rewiring of the three-dimensional (3D) genome architecture is increasingly being identified as the cause of genetic diseases, but recognizing the cis-regulatory effects of structural variation remains a challenge. The Xq27.1 region contains a quasi-palindrome identified as a pleiotropic hotspot for disease-causing interchromosomal insertions. In a large Danish family affected by X-linked recessive complex spastic paraplegia, we identified the segregation of a 149-kb interchromosomal insertion at Xq27.1 originating from 4q24. To understand the disease mechanism, we generated induced pluripotent stem cells (iPSCs) from affected individuals. Using CRISPR perturbation and neural differentiation experiments combined with high-throughput chromatin conformation capture (Hi-C) and transcriptomic analyses, we identify a 3D regulatory rewiring of SOX3 and transcriptional dysregulation of SOX3 targets in iPSC-derived neurons. Consistent with regulatory partitioning of the SOX3 topologically associating domain (TAD) in affected individuals, our experiments show that upstream cis-regulatory elements have a reduced ability to activate SOX3 expression and that the observed dysregulation depends on CTCF-binding sites within the insertion. This work provides mechanistic evidence that a position effect at the SOX3 locus can cause hereditary spastic paraplegia.
T. Terkelsen, V. Yumiceba, Joshua Kim et al.· American Journal of Human Ge...· 0 citations
Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor’s genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.
D. Skoczek, Jerzy Hohendorff, Maciej T. Małecki et al.· Human Genetics· 0 citations