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Ethylene (C2H4) is an important product in carbon dioxide electroreduction (CO2RR) because of the essential role it plays in chemical industry. While several strategies have been proposed to tune the selectivity of Cu-based catalysts in order to achieve high C2H4faradaic efficiency, maintaining high selectivity toward C2H4in CO2RR remains an unresolved problem hampering the deployment of CO2conversion technology due to the lack of stable electrocatalysts. Here, we develop a facile method to deposit a layer of Cu2O on Cu foil by an electrochemical pulsed potential treatment. This method is capable to easily scale up and synthesize multiple electrodes in one step. After the synthesis, the pulsed copper foil, denoted as P-Cu, exhibits good C2H4faradaic efficiency of ∼50% in CO2RR at a potential around −1.0 V vs. RHE. The C2H4selectivity is also found to be quantitatively correlated with the roughness factor (RF) of Cu-based catalysts. More importantly, for the first time, we demonstrate that the P-Cu electrode is quite durable in CO2RR to produce C2H4for more than 6 months.

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle1–3. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci4–10, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA–RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA–RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term ‘DR-loops’, which contain both DNA–DNA and DNA–RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops.

Organogenesis is crucial for proper organ formation during mammalian embryonic development. However, the similarities and shared features between different organs and the cellular heterogeneity during this process at single-cell resolution remain elusive. We perform single-cell RNA sequencing analysis of 1916 individual cells from eight organs and tissues of E9.5 to E11.5 mouse embryos, namely, the forebrain, hindbrain, skin, heart, somite, lung, liver, and intestine. Based on the regulatory activities rather than the expression patterns, all cells analyzed can be well classified into four major groups with epithelial, mesodermal, hematopoietic, and neuronal identities. For different organs within the same group, the similarities and differences of their features and developmental paths are revealed and reconstructed. We identify mutual interactions between epithelial and mesenchymal cells and detect epithelial cells with prevalent mesenchymal features during organogenesis, which are similar to the features of intermediate epithelial/mesenchymal cells during tumorigenesis. The comprehensive transcriptome at single-cell resolution profiled in our study paves the way for future mechanistic studies of the gene-regulatory networks governing mammalian organogenesis.

High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.

The development of the digestive tract is critical for proper food digestion and nutrient absorption. Here, we analyse the main organs of the digestive tract, including the oesophagus, stomach, small intestine and large intestine, from human embryos between 6 and 25 weeks of gestation as well as the large intestine from adults using single-cell RNA-seq analyses. In total, 5,227 individual cells are analysed and 40 cell types clearly identified. Their crucial biological features, including developmental processes, signalling pathways, cell cycle, nutrient digestion and absorption metabolism, and transcription factor networks, are systematically revealed. Moreover, the differentiation and maturation processes of the large intestine are thoroughly investigated by comparing the corresponding transcriptome profiles between embryonic and adult stages. Our work offers a rich resource for investigating the gene regulation networks of the human fetal digestive tract and adult large intestine at single-cell resolution. Gao et al. provide a comprehensive single-cell transcriptomic resource of four organs from the human fetal gastrointestinal tract and adult large intestine.

DNA methylation is a crucial layer of epigenetic regulation during mammalian embryonic development1–3. Although the DNA methylome of early human embryos has been analyzed4–6, some of the key features have not been addressed thus far. Here we performed single-cell DNA methylome sequencing for human preimplantation embryos and found that tens of thousands of genomic loci exhibited de novo DNA methylation. This finding indicates that genome-wide DNA methylation reprogramming during preimplantation development is a dynamic balance between strong global demethylation and drastic focused remethylation. Furthermore, demethylation of the paternal genome is much faster and thorough than that of the maternal genome. From the two-cell to the postimplantation stage, methylation of the paternal genome is consistently lower than that of the maternal genome. We also show that the genetic lineage of early blastomeres can be traced by DNA methylation analysis. Our work paves the way for deciphering the secrets of DNA methylation reprogramming in early human embryos.

Tin oxide based materials have been identified as active catalysts for electrochemical CO2-to-formate conversion. However, the oxide is unstable and can easily reconstruct via self-reduction under CO2reduction reaction (CO2RR) condition, resulting in undesired elevated hydrogen evolution reactivity. In this work, a stable tin dioxide (SnO2-1) electrocatalyst with abundant surface hydroxyls was synthesized via sodium cation assisted calcination method. The resulting electrocatalyst exhibits higher selectivity toward formate in a wide potential window compared with the one with very little hydroxyl groups. In addition, we demonstrate that SnO2-1 can operate at high current density of 200 mA·cm−2for at least 6 h while maintaining FEformateover 80%. Assisted with density functional theory (DFT) calculations, we propose that the presence of surface hydroxyls factors significantly into the overall performance of CO2RR, including optimization of the formation energy of key intermediate *OCHO and help SnO2to preserve partially reduced active surface.

TGF-β induces senescence in embryonic tissues. Whether TGF-β in the hypoxic tumor microenvironment (TME) induces senescence in cancer and how the ensuing senescence-associated secretory phenotype (SASP) remodels the cellular TME to influence immune checkpoint inhibitor (ICI) responses are unknown. We show that TGF-β induces a deeper senescent state under hypoxia than under normoxia; deep senescence correlates with the degree of E2F suppression and is marked by multinucleation, reduced reentry into proliferation, and a distinct 14-gene SASP. Suppressing TGF-β signaling in tumors in an immunocompetent mouse lung cancer model abrogates endogenous senescent cells and suppresses the 14-gene SASP and immune infiltration. Untreated human lung cancers with a high 14-gene SASP display immunosuppressive immune infiltration. In a lung cancer clinical trial of ICIs, elevated 14-gene SASP is associated with increased senescence, TGF-β and hypoxia signaling, and poor progression-free survival. Thus, TME-induced senescence may represent a naturally occurring state in cancer, contributing to an immune-suppressive phenotype associated with immune therapy resistance.

Chromatin remodeling is important for the epigenetic reprogramming of human primordial germ cells. However, the comprehensive chromatin state has not yet been analyzed for human fetal germ cells (FGCs). Here we use nucleosome occupancy and methylation sequencing method to analyze both the genome-wide chromatin accessibility and DNA methylome at a series of crucial time points during fetal germ cell development in both human and mouse. We find 116 887 and 137 557 nucleosome-depleted regions (NDRs) in human and mouse FGCs, covering a large set of germline-specific and highly dynamic regulatory genomic elements, such as enhancers. Moreover, we find that the distal NDRs are enriched specifically for binding motifs of the pluripotency and germ cell master regulators such as NANOG, SOX17, AP2γ and OCT4 in human FGCs, indicating the existence of a delicate regulatory balance between pluripotency-related genes and germ cell-specific genes in human FGCs, and the functional significance of these genes for germ cell development in vivo. Our work offers a comprehensive and high-resolution roadmap for dissecting chromatin state transition dynamics during the epigenomic reprogramming of human and mouse FGCs.

Circular RNAs (circRNAs) are a new class of non-polyadenylated non-coding RNAs that may play important roles in many biological processes. Here we develop a single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq) method to sequence both polyadenylated and non-polyadenylated RNAs from individual cells. This method exhibits robust sensitivity, precision and accuracy. We discover 2891 circRNAs and 913 novel linear transcripts in mouse preimplantation embryos and further analyze the abundance of circRNAs along development, the function of enriched genes, and sequence features of circRNAs. Our work is key to deciphering regulation mechanisms of circRNAs during mammalian early embryonic development.

The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution. Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These modifications enable scRRBS to provide digitized methylation information on ∼1 million CpG sites within an individual diploid mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements for DNA methylation. The entire procedure takes ∼3 weeks, and it requires strong molecular biology skills.

The epigenomes of mammalian sperm and oocytes, characterized by gamete-specific 5-methylcytosine (5mC) patterns, are reprogrammed during early embryogenesis to establish full developmental potential. Previous studies have suggested that the paternal genome is actively demethylated in the zygote while the maternal genome undergoes subsequent passive demethylation via DNA replication during cleavage. Active demethylation is known to depend on 5mC oxidation by Tet dioxygenases and excision of oxidized bases by thymine DNA glycosylase (TDG). Here we show that both maternal and paternal genomes undergo widespread active and passive demethylation in zygotes before the first mitotic division. Passive demethylation was blocked by the replication inhibitor aphidicolin, and active demethylation was abrogated by deletion of Tet3 in both pronuclei. At actively demethylated loci, 5mCs were processed to unmodified cytosines. Surprisingly, the demethylation process was unaffected by the deletion of TDG from the zygote, suggesting the existence of other demethylation mechanisms downstream of Tet3-mediated oxidation.

In the PDF version of this Resource originally published, on the author list the superscript number 9 to indicate Rui Wang was an equally contributing author was missing owing to a technical error. This has now been amended. In addition, the authors wish to replace Supplementary Table 2, as in the original version Group 1 immune cells were mis-classified into Group 46 immune cells, resulting in three groups of immune cells where there should have been four. Supplementary Table 2 has now been replaced.

Human fetal germ cells (FGCs) are precursors to sperm and eggs and are crucial for maintenance of the species. However, the developmental trajectories and heterogeneity of human FGCs remain largely unknown. Here we performed single-cell RNA-seq analysis of over 2,000 FGCs and their gonadal niche cells in female and male human embryos spanning several developmental stages. We found that female FGCs undergo four distinct sequential phases characterized by mitosis, retinoic acid signaling, meiotic prophase, and oogenesis. Male FGCs develop through stages of migration, mitosis, and cell-cycle arrest. Individual embryos of both sexes simultaneously contain several subpopulations, highlighting the asynchronous and heterogeneous nature of FGC development. Moreover, we observed reciprocal signaling interactions between FGCs and their gonadal niche cells, including activation of the bone morphogenic protein (BMP) and Notch signaling pathways. Our work provides key insights into the crucial features of human FGCs during their highly ordered mitotic, meiotic, and gametogenetic processes in vivo.

Active DNA demethylation in mammals involves ten-eleven translocation (TET) family protein-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC) (an oxidized derivative of 5mC) at the single-cell level remain unexplored. Here, we present “CLEVER-seq” (chemical-labeling-enabled C-to-T conversion sequencing), which is a single-cell, single-base resolution 5fC-sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq shows intrinsic 5fC heterogeneity in mouse early embryos, Epi stem cells (EpiSCs), and embryonic stem cells (ESCs). CLEVER-seq of mouse early embryos also reveals the highly patterned genomic distribution and parental-specific dynamics of 5fC during mouse early pre-implantation development. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells.