DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of ...homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.
Abstract Despite the advances in the understanding of reproductive physiology, the mechanisms underlying ovarian aging are still not deciphered. Recent research found an association between impaired ...ATM-mediated DNA double-strand break (DSB) repair mechanisms and oocyte aging. However, direct evidence connecting ATM-mediated pathway function decline and impaired oocyte quality is lacking. The objective of this study was to determine the role of ATM-mediated DNA DSB repair in the maintenance of oocyte quality in a mouse oocyte knockdown model. Gene interference, in vitro culture, parthenogenesis coupled with genotoxicity assay approaches, as well as molecular cytogenetic analyses based upon next-generation sequencing, were used to test the hypothesis that intact ATM function is critical in the maintenance of oocyte quality. We found that ATM knockdown impaired oocyte quality, resulting in poor embryo development. ATM knockdown significantly lowered or blocked the progression of meiosis in vitro, as well as retarding and reducing embryo cleavage after parthenogenesis. After ATM knockdown, all embryos were of poor quality, and none reached the blastocyst stage. ATM knockdown was also associated with an increased aneuploidy rate compared to controls. Finally, ATM knockdown increased the sensitivity of the oocytes to a genotoxic active metabolite of cyclophosphamide, with increased formation of DNA DSBs, reduced survival, and earlier apoptotic death compared to controls. These findings suggest a key role for ATM in maintaining oocyte quality and resistance to genotoxic stress, and that the previously observed age-induced decline in oocyte ATM function may be a prime factor contributing to oocyte aging.
CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed ...in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (
) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the
on-target locus, as well as segmental loss and gain of chromosome 6, on which the
gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.
Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use ...CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.
Mutations of the beta-globin gene (HBB) cause beta-thalassaemia and sickle cell anaemia. These are the most common cause of severe inherited disease in humans. Traditional preimplantation genetic ...testing protocols for detecting HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. We, therefore, asked the question whether a universally applicable preimplantation genetic testing method can be developed to test for HBB gene mutations.
A multiplex polymerase chain reaction protocol was designed, allowing simultaneous amplification of multiple overlapping DNA fragments encompassing the entire HBB gene sequence in addition to 17 characterized, closely linked single nucleotide polymorphisms (SNP). Amplicons were then analysed using a next-generation sequencing method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified DNA derived from embryos of three couples carrying different combinations of beta-thalassaemia mutations.
The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Analysis of 141 heterozygous sites showed no instances of allele dropout and the test displayed 100% concordance compared with the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive next-generation sequencing may provide superior accuracy than typically achieved using traditional preimplantation genetic testing methods. Importantly, no patient-specific test design or optimization was needed.
It is hoped that protocols that deliver almost universally applicable low-cost tests, without compromising diagnostic accuracy, will improve patient access to preimplantation genetic testing, especially in less affluent parts of the world.
Mutations of the beta-globin gene (HBB) are the most common cause of inherited disease in humans, causing ß-thalassaemia and sickle cell anaemia. Traditional preimplantation genetic testing (PGT) ...protocols for the detection of HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. Chapter 1 focuses on the development, validation and clinical implementation of a novel and universally applicable PGT method for the diagnosis of HBB gene mutations, utilising next generation sequencing (NGS). Employing this protocol HBB mutation status and associated single nucleotide polymorphism (SNP) haplotypes were successfully determined in all 21 embryos of three couples undergoing PGT for ß-thalassaemia. Analysis of 141 heterozygous sites showed no instances of allele dropout in the clinical samples and the test displayed 100% concordance compared with the data obtained using an established method (karyomapping). Taken together, the results suggest that the new method may deliver superior accuracy than typically achieved with traditional PGT methods. Furthermore, the test is streamlined and economical, which should improve patient access to PGT, reducing costs and waiting times. This will be especially important in less affluent parts of the world where diseases affecting hemoglobin synthesis are of high prevalence. An alternative to PGT for reducing the burden of inherited disorders is the correction of disease-causing mutations in human embryos using genome editing methods. However, this possibility is subject to numerous ethical and technical concerns, especially as intervention in gametes or preimplantation embryos would inevitably involve modification of the germline and a high likelihood of transmission of edited genes to future generations. CRISPR-Cas9 is currently the leading technology for introducing specific and heritable modifications into the genome. Chapter 2 evaluated the CRISPR-Cas9 system from the perspective of technical feasibility. It involved development of a methodological framework and computational pipelines for evaluation of on-target mutagenesis as well as potential off-target consequences of the editing in OCT4 (POU5F1) CRISPR-Cas9-targeted human zygotes and controls. A high efficiency of editing was observed with a wide variety of indel mutations, characteristic of non-homologous end-joining (NHEJ). No evidence of off-target activity was recorded. There is a possibility of unintended editing outcomes following the use of CRISPR-Cas9 that may have pathologic consequences, potentially exacerbated by insufficient DNA repair in early human embryos prior to embryonic genome activation (EGA). Chapter 3 investigated the repair outcomes of Cas9-induced double-strand breaks (DSBs) introduced in the POU5F1 locus. Strikingly, the results showed that 37.5% of the targeted zygotes present with breaks that remain unrepaired or participate in complex genomic rearrangements, resulting in segmental aneuploidy with breakpoints within the targeted 6p21.3 region. Gains and losses of large regions, stretching from 6p21.3 to the end of the short arm of chromosome 6, as well evidence indicating a complexity of DNA sequence mutations at on-target sites after CRISPR-Cas9 editing, provide a cautionary note to those considering the technology for clinical use and underscore the importance of basic research into DNA repair pathways and genomic stability in human embryos. Such research is not only relevant in the context of genome editing, but also has importance for assisted reproductive treatments and stem cell research. Furthermore, collaborative work of which this thesis is a part emphasizes that CRISPR-Cas9 remains a powerful molecular biology tool for the study of gene function and the biology of early human development.
Gene expression regulation is a complex and highly organized process involving a variety of genomic factors. It is widely accepted that differences in gene expression can contribute to the phenotypic ...variability between species, and that their interpretation can aid in the understanding of the physiologic variability. CNVs and miRNAs are two major players in the regulation of expression plasticity and may be responsible for the unique phenotypic characteristics observed in different lineages. We have previously demonstrated that a close interaction between these two genomic elements may have contributed to the regulation of gene expression during evolution. This work presents the molecular interactions between CNV and non CNV genes with miRNAs and other genomic elements in eight different species. A comprehensive analysis of these interactions indicates a unique nature of human CNV genes regulation as compared to other species. By using genes with short 3' UTR that abolish the "canonical" miRNA-dependent regulation, as a model, we demonstrate a distinct and tight regulation of human genes that might explain some of the unique features of human physiology. In addition, comparison of gene expression regulation between species indicated that there is a significant difference between humans and mice possibly questioning the effectiveness of the latest as experimental models of human diseases.
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