Dieter Egli

In early mammalian embryo development, DNA replication stress can lead to fork collapse, double-strand breaks, chromosome breakage, aneuploidy, and developmental failure. However, the causes and consequences of DNA replication stress remain poorly understood. Here, we show that DNA replication stress in the early embryo, characterized by slow fork speed and γH2AX foci in G2 phase, is alleviated in part by nucleoside supplementation, pointing to limiting replication factors as a cause of this stress; DNA demethylation, by contrast, plays no role in fork speed or γH2AX formation. We further show that ssDNA gaps form in both mouse and human preimplantation embryos during cleavage divisions, in a PrimPol-dependent manner, and give rise to γH2AX foci. These gaps are filled by the translesion polymerase Rev1, and delay mitotic entry; premature entry into mitosis with unresolved gaps causes DNA breakage, foremost in late-replicating regions, impairing developmental potential. Finally, we show that ssDNA gaps are vulnerable to unspecific, gRNA-independent base editor activity. Together, these findings identify ssDNA gaps as a source of chromosome breakage and aneuploidy in the early embryo, and as a previously unrecognized target of base editors, with direct relevance to both the basic biology of reproduction and the safety of germline gene editing.

DNA repair in human embryos is poorly understood, and double-strand breaks (DSBs) can cause chromosome loss. We show that chromosomal alterations relative to an induced DSB are asymmetric: acentric arms show complementary gains and losses, while centric arms are biased toward losses. Centromeric to the cut site secondary breakage and attrition is extensive. In contrast, break sites at acentric arms are conserved with no secondary breakage. These differences reflect differential forces at the mitotic spindle. Telomeric arms detach from the pro-metaphase spindle while centric truncated chromosomes lag during anaphase, suggesting that the DSB impedes sister chromatid separation. Secondary breakage near the centromere concordant with extensive attrition at the DSB site indicates a DSB can destabilize a chromosome without end-joining of sister chromatids. These results highlight the risks of chromosomal-scale changes in CRISPR-Cas9 genome editing and show that a single DSB can destabilize a human embryo chromosome independent of fusion-breakage cycles.

Cas9-based tools enable the introduction of genetic lesions to investigate DNA repair outcomes and edit the genome at disease-relevant loci. DNA double-strand breaks (DSBs) induced by CRISPR/Cas9 result in frequent aneuploidy and large deletions, revealing a repair deficiency in early human embryos and limiting the clinical application of this technology. Here we evaluated the DNA repair outcomes of DNA nicks and mismatches introduced using base editors in human embryos at two targets, PCSK9 and HBG . Editing was efficient and, unlike Cas9-induced DSBs, did not result in either chromosomal abnormalities or large deletions. Small insertions or deletions after base editing were rare, and off-target activity was dependent on the guide RNA. Delivering the base editor as a protein at fertilization or at the pronuclear stage allowed normal development to the blastocyst stage and the derivation of edited stem cell lines. In stark contrast, introduction of the editor as RNA resulted in early embryo arrest. Our results demonstrated that, unlike DSBs, DNA nicks and mismatches are efficiently repaired in human embryos, allowing specific on-target changes without genotoxic consequences.

Stem cell–derived β cells offer a promising approach for type 1 diabetes (T1D) treatment. However, the processes of graft infiltration and rejection by immune cells remain poorly understood in humans. In this study, autologous or allogeneic stem cell–derived islets (SC-islets) were transplanted in human immune system mice and analyzed 14 to 18 weeks later. Imaging mass cytometry revealed unique characteristics of SC-islet grafts, including a high percentage of glucagon ⁺ cells and the presence of cysts and CD57 ⁺ enterochromaffin cells, features not typically observed in endogenous or transplanted allogeneic primary pancreatic islets. Allogeneic SC-islet grafts exhibited heavy immune infiltration, cell proliferation, and pro-fibrotic processes, whereas autologous grafts showed minimal infiltration and little fibrosis. In some mice, autologous T cells expressing islet antigen-reactive (IAR) T cell receptors (TCRs) were adoptively transferred. Three weeks after transfer, autologous grafts injected with IAR-TCR ⁺ T cells showed negligible immune infiltration, even though IAR-TCR ⁺ T cells were detected in the spleen. Under the conditions tested, human SC-islet grafts were not rejected by an autologous immune system, even in the presence of autoreactive T cells, pointing to several limitations that remain to be addressed for a model of spontaneous autologous SC-islet infiltration and destruction.

(1) Aims and hypothesis: Loss-of-function mutations in SLC30A8 , encoding the zinc ion (Zn ²⁺ ) transporter ZnT8 in pancreatic beta cells, lower type 2 diabetes risk dose-dependently, but the underlying mechanisms remain unclear. Here, we combine proteomic, transcriptomic and functional approaches in human stem cell-derived islet-like clusters bearing common alleles or the inactivating variant R138X. We hypothesized that this variant protects against the deleterious effect of Zn ²⁺ depletion on cell survival and function. (2) Methods: Human embryonic stem cells INS(GFP/w) (MEL1), and CRISPR/Cas9-derived heterozygous or homozygous R138X lines were differentiated into stem cell-derived islet-like clusters. Intracellular Zn ²⁺ levels were reduced using the chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-1,2-ethanediamine (TPEN). Apoptosis was assessed by TUNEL staining and protein expression by immunofluorescence. Glucose-stimulated calcium (Ca ²⁺ ) dynamics were measured using the intracellular probe (Cal590) and insulin secretion by homogenous time-resolved fluorescence. Transcriptomic profiling was performed by bulk mRNA sequencing and proteomics by liquid chromatography-tandem mass spectrometry. (3) Results: Intracellular Zn ²⁺ depletion increased apoptosis in wild-type islet-like clusters, whereas R138X clusters were protected. R138X heterozygous clusters showed a mild increase in GCG ⁺ cells and R138X homozygous clusters exhibited increased NKX6.1 ⁺ cells, without affecting polyhormonal populations. These changes were reversed under Zn ²⁺ depletion. Transcriptomic and proteomic analyses, assessing genotype effects while accounting for Zn ²⁺ depletion, showed that R138X clusters (versus wild-type) exhibited upregulation of genes and proteins involved in vesicle trafficking, secretion, Ca²⁺ signaling and mitochondrial metabolism, consistent with enhanced glucose-stimulated insulin secretion in homozygous clusters. Conversely, genes and proteins associated with extracellular matrix remodeling, metal-ion handling, apoptosis and cellular stress were downregulated. R138X clusters displayed altered Ca ²⁺ signaling, with decreased area under the curve and oscillation amplitude, but increased frequency. These differences were reversed by TPEN, while Zn ²⁺ depletion impaired Ca ²⁺ response in wild-type clusters. Despite lowered overall activity, R138X homozygous clusters showed enhanced overall cell-cell connectivity, reversed by TPEN treatment. The opposite effects were observed in R138X heterozygous clusters, showing improved connectivity and activity under Zn ²⁺ depletion. (4) Conclusion and interpretation: Intracellular Zn ²⁺ depletion compromises islet-like cluster identity and function, while the R138X variant confers protection against these effects. Under Zn ²⁺ -depleted conditions, ZnT8 deficiency promotes a more mature and metabolically active state of the R138X clusters, with enhanced Ca ²⁺ signaling and insulin secretion, supported by a structural remodeling and the downregulation of apoptosis and cellular stress. These findings highlight the therapeutic potential of targeting ZnT8 in type 2 diabetes and support its relevance for further improving cell-based therapies. Research in Context: What is already know about this subject?: Rare inactivating mutations in the insulin granule-associated zinc transporter gene, SLC30A8/ZnT8 , drive lowered type 2 diabetes risk. Previous studies have indicated that apoptosis is lowered, and glucose-stimulated insulin secretion enhanced, after ZnT8 inactivation. The molecular mechanisms underlying these changes are unclear. What is the key question?: How do inactivating mutations in SL30A8/ZnT8 lead to lowered apoptosis and enhanced insulin secretion from stem cell-derived islet-like clusters, and is altered susceptibility to intracellular zinc depletion involved? What are the new findings?: The rare inactivating R138X mutation in SLC30A8 leads to gene dose-dependent changes in the transcriptome and proteome of islet-like clusters. Changes include upregulation of maturity and downregulation of immaturity genes. Depletion of intracellular Zn ²⁺ exaggerates the protective effects of the inactivating mutation on apoptosis and insulin secretion How might this impact on clinical practice in the foreseeable future?: Our findings suggest that careful monitoring of both dietary zinc intake and of circulating levels of zinc ions, whose effects are mitigated in SLC30A8 mutation carriers, may be helpful in some populations to lower diabetes risk.

Transcriptional regulatory elements (TREs) orchestrate gene expression programs fundamental to cellular identity and transitions across physiological and pathological states. Here, we present a high-resolution atlas of RNA Polymerase II-engaged TREs (enhancers and promoters) across all major human organ systems and a broad spectrum of developmental and disease states. This atlas is generated using PRO-cap, a highly sensitive method that detects nascent RNA at transcription initiation sites, a critical feature of active TREs. The base-pair resolution of PRO-cap enables systematic dissection of transcription initiation architecture, revealing associations among tissue specificity, evolutionary constraint, transcription factor usage, and regulatory connectivity. Integration with deep learning models such as ProCapNet further provides a framework for prioritizing noncoding variants from GWAS and eQTL studies. Moreover, this tissue-resolved atlas identifies lineage-specific regulatory programs and their alterations in diseases such as metastatic cancer, where TRE landscapes capture regulatory signatures reflecting both tissue of origin and adaptive responses to distant niches. Together, these findings establish transcription initiation at regulatory elements as a defining and mechanistically informative layer of gene regulation across development, physiology, and disease.

Mammalian species exist as diploid organisms, with only gametes existing as stable, nonproliferating haploids. Haploid mammalian pluripotent stem cells, in all derived species to date - mouse, human and rat - undergo spontaneous diploidization. Here, we investigated the mechanisms affecting the stability of the haploid state using the mouse and human embryo, as well as a human embryonic stem cells. We demonstrate that diploidization occurs early in preimplantation development and is often unproductive, with haploid embryos exhibiting decreased developmental potential. Haploid embryos show increased chromosome segregation errors at the first mitosis, failure to align chromosomes on the metaphase plate in the second mitosis. Delayed mitotic progression and failure to form a central spindle and a midbody are followed by cytokinesis failure, diploidization, increased DNA damage marked by γH2AX and RPA32 foci, and low developmental potential. By increasing the nucleo-cytoplasmic ratio in diploids, or reducing this ratio in haploids, these phenotypes can be induced in diploids or reduced in haploids. Changing ploidy alone from haploid to diploid without also adjusting nucleo-cytoplasmic ratio does not improve mitotic phenotypes and developmental outcomes. Thus, the most upstream driver responsible for the instability of the haploid state is a stage- specific ratio of nucleus to cell size.

Cas9 provides a powerful tool to interrogate DNA repair and to introduce targeted genetic modifications. However, a major challenge of Cas9-based editing in human embryos is the occurrence of chromosomal abnormalities caused by Cas9 cleavage. Furthermore, mosaicism - different genetic outcomes in different cells, prevent accurate genotyping using a single embryo biopsy. Through timed analysis of editing outcomes during the first cell cycle and timed inhibition of Cas9 using AcrIIA4, we show that most edits occur at least 12 hours post Cas9 injection and therefore after the first S-phase. This timing limits the ability to achieve uniform editing across cells. We found that segmental chromosomal abnormalities and the consequential loss of heterozygosity are common at Cas9 cleavage sites throughout the genome, including at MYBPC3 and at CCR5 loci, for which this has not previously been reported. Surprisingly, inhibiting Cas9 activity 8-12 hours before mitosis does not eliminate chromosomal aneuploidies. This suggests that double-strand break (DSB) repair in human embryos is exceedingly slow, with breaks remaining unrepaired for many hours. Thus, the timing of DSB induction and repair relative to the first S-phase and the first mitosis is intrinsically limiting to preventing mosaicism and maintaining genome stability in embryonic gene editing.