The correction of disease-causing mutations in human embryos could reduce the burden of inherited genetic disorders in the fetus and newborn, and improve the efficiency of fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here we evaluate the repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frame-shift 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. However, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, upon entry into mitosis, loss of one or both chromosomal arms. Thus, Cas9 allows for the modification of chromosomal content in human embryos in a targeted manner, which may be useful for the prevention of trisomies.

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Highlights d Generation of human androgenetic and parthenogenetic ESCs (aESCs and pESCs) d Comparing aESCs and pESCs identifies known and formerly undescribed imprinted genes d The uniparental cells show tissue-specific parent-of-origin differentiation biases d The imprinted gene IGF2 is involved in hepatic differentiation bias of human aESCs

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Many human diseases have an underlying genetic component. The development and application of methods to prevent the inheritance of damaging mutations through the human germline could have significant health benefits, and currently include preimplantation genetic diagnosis and carrier screening. Ma et al. take this a step further by attempting to remove a disease mutation from the human germline through gene editing 1 . They assert the following advances: (i) the correction of a pathogenic gene mutation responsible for hypertrophic cardiomyopathy in human embryos using CRISPR-Cas9 and (ii) the avoidance of mosaicism in edited embryos. In the case of correction, the authors conclude that repair using the homologous chromosome was as or more frequent than mutagenic nonhomologous end-joining (NHEJ). Their conclusion is significant, if validated, because such a "self-repair" mechanism would allow gene correction without the introduction of a repair template. While the authors' analyses relied on the failure to detect mutant alleles, here we suggest approaches to provide direct evidence for interhomologue recombination and discuss other events consistent with the data. We also review the biological constraints on inter-homologue recombination in the early embryo.In their first approach, Ma et al. used donor sperm from a patient heterozygous for the MYBPC3 ΔGAGT mutation to fertilize wild-type oocytes, such that half of the embryos started out as wild type at the MYBPC3 locus and half heterozygous. Fertilized zygotes were injected with Cas9 and an sgRNA directed to create a double-strand break (DSB) in the mutant paternal allele. The authors report that 24% of the embryos at day 3 of development were mosaic, with some cells of the embryo containing the mutant paternal locus, either intact or modified by NHEJ, together with a wild-type locus. Remaining cells of the embryo contained only a detectable wild-type allele. While some zygotes were also co-injected with a wild-type, exogenous, single-stranded oligodeoxynucleotide template (ssODN) with two synonymous mutations, no mutations consistent with ssODN-templated repair were detected. Furthermore, 'wild-type only' cells were present at a similar frequency both in the presence and absence of the ssODN. The authors infer that these cells arose by homology-directed repair (HDR) of the mutant paternal allele using the wild-type maternal allele as a template, i.e., inter-homologue recombination, leading to gene correction.In a second approach, earlier, MII-phase oocytes were coinjected with Cas9 complexes and donor sperm. In this case, mosaicism was not detected, except in a single embryo, which contained both 'wildtype only' cells and ones heterozygous for wild-type and ssODN-templated alleles. Although wild-type embryos were expected at 50% frequency, they appeared to comprise 72% of embryos.

␤-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, ␤-carotene 15,15-oxygenase (BCO1) converts ␤-carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitamin A that acts as a transcription factor ligand to regulate gene expression. ␤-Carotene can also be cleaved by ␤-carotene 9,10-oxygenase (BCO2) to form ␤-apo-10-carotenal, a precursor of retinoic acid and a transcriptional regulator per se. The mammalian embryo obtains ␤-carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that ␤-carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show that ␤-carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that ␤-apo-10-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4␣ and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of ␤-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that ␤-carotene induces a feed-forward mechanism in the placenta to enhance the assimilation of ␤-carotene for proper embryogenesis.The importance of vitamin A as a critical modulator of mammalian embryonic development has been known for decades (1). This essential nutrient exerts its function mainly through its active form, retinoic acid. Retinoic acid binds to retinoic acid receptors and retinoid X receptors (RXRs) 3 and regulates, in a spatial and temporal manner, the transcription of numerous genes vital to development (2-6).The mammalian embryo obtains retinoids (vitamin A and its derivatives) and provitamin A carotenoids from the maternal bloodstream. Among dietary carotenoids, ␤-carotene (BC) is the main source of vitamin A for the majority of the world population (7). Intact BC from the maternal circulation crosses the placenta and reaches the developing embryo where the cytoplasmic ␤-carotene 15,15Ј-oxygenase (BCO1) cleaves BC symmetrically to yield retinaldehyde, which in turn is oxidized to retinoic acid (8, 9). Asymmetric cleavage of BC by ␤-carotene 9Ј,10Ј-oxygenase (BCO2) also occurs, generating ␤-ionone and ␤-apo-10Ј-carotenal (apo10AL) (9). The latter, as well as other ␤-apocarotenoids of various chain lengths generated from oxidative breakdown of BC in food and animal tissues (10), can be converted into one molecule of retinaldehyde by BCO1 (9, 11). H...

The correction of disease-causing mutations in human embryos could reduce the burden of inherited genetic disorders in the fetus and newborn, and improve the efficiency of fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here we evaluate the repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frame-shift 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. However, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, upon entry into mitosis, loss of one or both chromosomal arms.Thus, Cas9 allows for the modification of chromosomal content in human embryos in a targeted manner, which may be useful for the prevention of trisomies.

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 G 1 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 G 1 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.