Measuring gene expression in individual cells is crucial for understanding the gene regulatory network controlling human embryonic development. Here we apply single-cell RNA sequencing (RNA-Seq) analysis to 124 individual cells from human preimplantation embryos and human embryonic stem cells (hESCs) at different passages. The number of maternally expressed genes detected in our data set is 22,687, including 8,701 long noncoding RNAs (lncRNAs), which represents a significant increase from 9,735 maternal genes detected previously by cDNA microarray. We discovered 2,733 novel lncRNAs, many of which are expressed in specific developmental stages. To address the long-standing question whether gene expression signatures of human epiblast (EPI) and in vitro hESCs are the same, we found that EPI cells and primary hESC outgrowth have dramatically different transcriptomes, with 1,498 genes showing differential expression between them. This work provides a comprehensive framework of the transcriptome landscapes of human early embryos and hESCs.

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient1, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders2. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3,4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

More than 10,000 monogenic inherited disorders have been identified, affecting millions of people worldwide. Among these are autosomal dominant mutations, where inheritance of a single copy of a defective gene can result in clinical symptoms. Genes in which dominant mutations manifest as late-onset adult disorders include BRCA1 and BRCA2, which are associated with a high risk of breast and ovarian cancers 1 , and MYBPC3, mutation of which causes hypertrophic cardiomyopathy (HCM) 2 . Because of their delayed manifestation, these mutations escape natural selection and are often transmitted to the next generation. Consequently, the frequency of some of these founder mutations in particular human populations is very high. For example, the MYBPC3 mutation is found at frequencies ranging from 2% to 8% 3 in major Indian populations, and the estimated frequency of both BRCA1 and BRCA2 mutations among Ashkenazi Jews exceeds 2% 4 .HCM is a myocardial disease characterized by left ventricular hypertrophy, myofibrillar disarray and myocardial stiffness; it has an estimated prevalence of 1:500 in adults 5 and manifests clinically with heart failure. HCM is the commonest cause of sudden death in otherwise healthy young athletes. HCM, while not a uniformly fatal condition, has a tremendous impact on the lives of individuals, including physiological (heart failure and arrhythmias), psychological (limited activity and fear of sudden death), and genealogical concerns. MYBPC3 mutations account for approximately 40% of all genetic defects causing HCM and are also responsible for a large fraction of other inherited cardiomyopathies, including dilated cardiomyopathy and left ventricular non-compaction 6 . MYBPC3 encodes the thick filament-associated cardiac myosin-binding protein C (cMyBP-C), a signalling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of both contraction and relaxation 2 .Current treatment options for HCM provide mostly symptomatic relief without addressing the genetic cause of the disease. Thus, the development of novel strategies to prevent germline transmission of founder mutations is desirable. One approach for preventing second-generation transmission is preimplantation genetic diagnosis (PGD) followed by selection of non-mutant embryos for transfer in the context of an in vitro fertilization (IVF) cycle. When only one parent carries a heterozygous mutation, 50% of the embryos should be mutationfree and available for transfer, while the remaining carrier embryos are discarded. Gene correction would rescue mutant embryos, increase the number of embryos available for transfer and ultimately improve pregnancy rates.Recent developments in precise genome-editing techniques and their successful applications in animal models have provided an option for correcting human germline mutations. In particular, CRISPR-Cas9 is a versatile tool for recognizing specific genomic sequences and inducing DSBs 7-10 . DSBs are then resolved by endogenous DNA repair mechanisms, prefer...

SUMMARY Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research.