This study provides a robust framework for modeling human-specific regulatory disorders and demonstrates the critical impact of non-coding variation on disease pathogenesis.
Abstract
Hirschsprung disease (HSCR) is a complex developmental disorder of the enteric nervous system, primarily driven by regulatory variants within enhancer elements of the RET gene. To investigate how these variants lead to aganglionosis, we developed a humanized mouse model by inserting an intact 77kb human RET genomic locus into the Rosa26 safe-harbor locus. Utilizing “big DNA” synthetic biology and Bxb1-mediated recombination, we integrated the complete human locus including all exons, introns, and upstream regulatory elements which we validated via nanopore and short-read sequencing. Functional analysis confirmed in vivo human RET expression; however, our initial HSCR-associated “sensitive” haplotype expressed at only 21% of wild-type levels. This significant reduction proved insufficient to rescue the viability when endogenous mouse Ret was deleted. We identified that this deficiency is partially driven by five risk SNPs within established enhancers. Specifically, using CRISPR/Cas9 to restore a conserved So×10 binding site (converting a sensitive SNP to a protective one) increased RET expression by 1.9-fold and restored transcription factor binding. This study provides a robust framework for modeling human-specific regulatory disorders and demonstrates the critical impact of non-coding variation on disease pathogenesis.
This work provides mechanistic evidence that a position effect at the SOX3 locus can cause hereditary spastic paraplegia and identifies a 3D regulatory rewiring of SOX3 and transcriptional dysregulation of SOX3 targets in iPSC-derived neurons.
T. Terkelsen, V. Yumiceba, Joshua Kim et al.· American Journal of Human Ge...· 0 citations
The results identify Pgm3 as the most promising candidate for further development as a Ciona-based model of human disease and demonstrate the utility of tissue-specific CRISPR screening for prioritizing candidate disease gene orthologs identified through comparative genomics platforms like Zoogle.
Sabrina A. Hernandez, Christopher J. Johnson, Alberto Stolfi· bioRxiv· 0 citations
This study provides direct evidence that VEXAS-specific TE govern HSC clonal dominance, thereby uncovering a regulatory axis underlying HSC biology and disease mechanisms, opening a therapeutic strategy directed towards the repetitive genome.
A. Varesi, Sontago Dong, Chiara Gaddoni et al.· bioRxiv· 0 citations
Gene editing in livestock using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) offers a promising approach for genetic improvement in cattle. This study evaluated germline transmission and mutation stability of double-knockout cattle generated by zygote electroporation. Previously reported myostatin/beta-lactoglobulin (MSTN/BLG) and newly generated α-1,3-galactosyltransferase (GGTA1/BLG) double-knockout cattle were produced using CRISPR/Cas9-mediated genome editing. Targeted deep sequencing demonstrated extensive somatic mosaicism across multiple tissues. Computer-assisted sperm analysis (CASA) demonstrated normal sperm motility in MSTN/BLG double-knockout males. Fertilization of wild-type oocytes produced heterozygous embryos, with mutation frequencies of 37.76 ± 10.74% at the MSTN locus and 54.80 ± 7.73% at the BLG locus, as assessed by T7 endonuclease I (T7E1) assay. MSTN/BLG double-knockout sperm were subsequently used for embryo production and for artificial insemination of GGTA1/BLG double-knockout females. Healthy offspring were successfully obtained (n = 3), alongside one stillborn calf. Targeted deep sequencing of all four progenies revealed highly variable allele frequencies that deviated substantially from the approximately 50% expected for heterozygous germline transmission. In contrast, whole-genome sequencing (WGS) results were consistent with Mendelian expectations, underscoring the limitations of PCR-based targeted sequencing for assessing germline transmission in mosaic founders. These results show that CRISPR/Cas9-edited embryos generated by electroporation can develop into healthy, sexually mature cattle capable of germline transmission. While variable transmission rates were observed owing to founder mosaicism, non-mosaic F1 offspring were successfully generated. However, direct, embryo-mediated gene-editing strategies remain technically and economically challenging for large-scale commercial calf production, and reports in cattle are limited. This study provides a reference for future applications of gene-edited embryos and their germline propagation.
Kyeong-Hyeon Eom, G. Gim, Min-Gyu Lee et al.· Theriogenology· 0 citations
In mammalian cells, DNA double-strand breaks (DSBs) are repaired by two competing pathways-homologous recombination (HR) and non-homologous end-joining (NHEJ)-that act on the same DNA ends. Downregulation of NHEJ has been shown to enhance HR-mediated repair. Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by progressive corneal opacity and vision loss in humans. More than 180 mutations in the CHST6 gene are linked to MCD, with over 70% occurring in exon 3, making it a promising target for genome editing. In this study, we performed in vitro editing of exon 3 of CHST6 using CRISPR/Cas9 in human embryonic kidney (HEK293) cells. To promote HR, the NHEJ genes XRCC6 and XRCC5, encoding KU70 and KU80, were knocked down individually or in combination. A homologous donor template was also introduced, and HR efficiency was assessed by Western blot analysis. Results demonstrated a significant increase in HR activity following downregulation of these NHEJ components, as indicated by elevated RAD51 expression. As proof of concept, partial restoration of CHST6 protein expression was observed in edited cells compared with CHST6 knockdown controls after suppression of XRCC6 and XRCC5 along with donor template delivery. These findings suggest that targeting NHEJ to enhance HR may represent a promising therapeutic strategy for MCD.
Aparajita Baruah, Tobias Wimmer, K. Stieger et al.· Experimental Eye Research· 0 citations
It is reported that the choice of sgRNA target site affects its targeted cleavage activity and show that the combination of guideRNA with SpCas9 can effectively delete the exonic fragment of the SLC25A38 gene in HEK293T cells.
S. Yazdanparast, Hamid Galehdari, S. Khatami et al.· Journal of Applied Genetics· 0 citations
