Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing ...mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
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•Cas9-mediated DSB induction and repair by end joining occurs within hours•End joining provides an efficient way to restore reading frames without mosaicism•Unrepaired DSBs persist through mitosis and result in frequent chromosome loss•Off-target effects of Cas9 cause indels as well as chromosome loss
CRISPR-Cas9 gene editing in early human embryos leads to frequent loss of the targeted chromosome, indicating that human germline gene editing would pose a substantial risk for aneuploidy and other adverse genetic consequences
Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low ...fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential.
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•1-cell embryos show replication fork stalling, with replication extending into G2 phase•Incompletely replicated DNA is converted to chromosome breaks and aneuploidy in mitosis•Spontaneous chromosome breaks and G2 DNA synthesis occur in congruent gene-poor regions•Chromosome fragility in human embryos occurs independently of embryonic genome activation
In human preimplantation embryos, DNA replication in G2 phase results in chromosome breakage, segmental aneuploidies, and poor embryo quality.
Genomic imprinting is an epigenetic mechanism that results in parent-of-origin monoallelic expression of specific genes, which precludes uniparental development and underlies various diseases. Here, ...we explored molecular and developmental aspects of imprinting in humans by generating exclusively paternal human androgenetic embryonic stem cells (aESCs) and comparing them with exclusively maternal parthenogenetic ESCs (pESCs) and bi-parental ESCs, establishing a pluripotent cell system of distinct parental backgrounds. Analyzing the transcriptomes and methylomes of human aESCs, pESCs, and bi-parental ESCs enabled the characterization of regulatory relations at known imprinted regions and uncovered imprinted gene candidates within and outside known imprinted regions. Investigating the consequences of uniparental differentiation, we showed the known paternal-genome preference for placental contribution, revealed a similar bias toward liver differentiation, and implicated the involvement of the imprinted gene IGF2 in this process. Our results demonstrate the utility of parent-specific human ESCs for dissecting the role of imprinting in human development and disease.
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•Generation of human androgenetic and parthenogenetic ESCs (aESCs and pESCs)•Comparing aESCs and pESCs identifies known and formerly undescribed imprinted genes•The uniparental cells show tissue-specific parent-of-origin differentiation biases•The imprinted gene IGF2 is involved in hepatic differentiation bias of human aESCs
Benvenisty, Egli, and colleagues combine newly derived human androgenetic ESCs with parthenogenetic and bi-parental ESCs to explore parental imprinting in humans, charting the regulatory landscape of known imprinted loci, identifying previously undescribed imprinted genes, and interrogating the nature and mechanisms underlying parental-origin-driven tissue-specific differentiation biases with implications for human development and disease.
Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we ...profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.
Abstract DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, ...that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.
Reprogrammed pluripotent stem cells (PSCs) are valuable for research and potentially for cell replacement therapy. However, only a fraction of reprogrammed PSCs are developmentally competent. Genomic ...stability and accurate DNA synthesis are fundamental for cell development and critical for safety. We analyzed whether defects in DNA replication contribute to genomic instability and the diverse differentiation potentials of reprogrammed PSCs. Using a unique single-molecule approach, we visualized DNA replication in isogenic PSCs generated by different reprogramming approaches, either somatic cell nuclear transfer (NT-hESCs) or with defined factors (iPSCs). In PSCs with lower differentiation potential, DNA replication was incompletely reprogrammed, and genomic instability increased during replicative stress. Reprogramming of DNA replication did not correlate with DNA methylation. Instead, fewer replication origins and a higher frequency of DNA breaks in PSCs with incompletely reprogrammed DNA replication were found. Given the impact of error-free DNA synthesis on the genomic integrity and differentiation proficiency of PSCs, analyzing DNA replication may be a useful quality control tool.
Purpose
To correct a potentially damaging mutation in haploid human embryonic stem cells.
Methods
Exome sequencing was performed on DNA extracted from parthenogenetically derived embryonic stem cell ...line (pES12). An SLC10A2 gene mutation, which affects bile acid transport, was chosen as mutation of interest in this proof of concept study to attempt correction in human pluripotent haploid cells. Confirmation of the mutation was verified, and guide RNA and a correction template was designed in preparation of performing CRISPR. Haploid cells underwent serial fluorescence activated cell sorting (FACS) with Hoechst 33342 to create an increasingly haploid (1n) enriched culture. Nucleofection was performed on p. 37 and then cells were sorted for 1n DNA content with +GFP to identify the haploid cells that expressed Cas9 tagged with GFP.
Results
104,686 haploid GFP + cells were collected. Cells were cultured, individual colonies picked, and 48 clones were sent for Sanger sequencing. CRIPSR efficiency was 77.1%, with 7/48 (14.6%) clones resulting in a corrected SLC10A2 mutation. Confirmation of persistence of haploid cells was achieved with repeated FACS sorting and centromere quantification. Given the large number of passages and exposure to CRISPR, we also performed analysis of karyotypes and of off-target effects. Cells evaluated were karyotypically normal and there was no evident off target effects.
Conclusions
CRISPR/Cas9 can be effectively utilized to edit mutations in haploid human embryonic stem cells. Establishment and maintenance of a haploid cell culture provides a novel way to utilize CRISPR/Cas9 in gene editing, particularly in the study of recessive alleles.
The goal of this paper is to aggregate information on monogenic contributions to obesity in the past five years and to provide guidance for genetic testing in clinical care.
Advances in sequencing ...technologies, increasing awareness, access to testing, and new treatments have increased the utilization of genetics in clinical care. There is increasing recognition of the prevalence of rare genetic obesity from variants with mean allele frequency < 5% -new variants in known genes as well as identification of novel genes- causing monogenic obesity. While most of these genes are in the leptin melanocortin pathway, those in adipocytes may also contribute. Common variants may contribute either to higher lifetime tendency for weight gain or provide protection from monogenic obesity. While specific genetic mutations are rare, these segregate in individuals with early-onset severe obesity; thus, collectively genetic etiologies are not as rare. Some genetic conditions are amenable to targeted treatment. Research into the discovery of novel genetic causes as well as targeted treatment is growing over time. The utility of therapeutic strategies based on the genetic risk of obesity is an advancing frontier.
Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have ...unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.
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