Dear Editor, Large-scale phenotyping efforts have shown that placental defects correlate strongly with embryonic lethal mouse mutants(Perez-Garcia et al.,2018).To rescue normal development of these mutant embryos,tetraploid complemen-tation can be a reliable strategy,in which,tetraploid cells form extraembryonic lineages thus providing the embryo with a wild-type placenta(Nagy et al.,1990).

Genomic imprinting is required for sexual reproduction and embryonic development of mammals, in which, differentially methylated regions (DMRs) regulate the parent-specific monoallelic expression of imprinted genes. Numerous studies on imprinted genes have highlighted their critical roles in development. However, what imprinting network is essential for development is still unclear. Here, we establish a stepwise system to reconstruct a development-related imprinting network, in which diploid embryonic stem cells (ESCs) are derived by fusing between parthenogenetic (PG)- and androgenetic (AG)-haploid embryonic stem cells (haESCs) with different DMR deletions (termed Ha-Ha-fusion system), followed by tetraploid complementation to produce all-haESC fetuses. Diploid ESCs fused between PG-haESCs carrying 8 maternally-derived DMR deletions and AG-haESCs with 2 paternally-derived DMR deletions give rise to live pups efficiently, among which, one lives to weaning. Strikingly, diploid ESCs derived from the fusion of PG-haESCs with 7 maternal DMR deletions and AG-haESCs with 2 paternal DMR deletions and maternal Snrpn-DMR deletion also support full-term embryonic development. Moreover, embryos reconstructed by injection of AG-haESCs with hypomethylated H19-DMR into oocytes with H19-DMR deletion develop into live mice sustaining inverted allelic gene expression. Together, our findings indicate that restoration of monoallelic expression of 10 imprinted regions is adequate for the full-term development of all-haESC pups, and it works irrespective of their parental origins. Meanwhile, Ha-Ha-fusion system provides a useful tool for deciphering imprinting regulation networks during embryonic development.

Chromosomes are spatially organized and functionally folded into a specific macro-structure in the nucleus. Recently, we and others created haploid cells with chromosome fusions. However, there is still lack of an effective strategy for precisely investigating how the genome copes with fusions. Here, we developed a down-sampling method to convert the populational Hi-C dataset into single cell-like Khimaira Matrix (K-matrix). K-matrix preserves not only the most prominent functional genomic features but also cell-to-cell variations. K-matrix-originated genome 3D models display spatial approach of fused chromosomes and minor global structure alterations. Combined with a layered positional decomposition analysis, our models indicate slight re-adjustment of chromosome distributions accordingly with an increasing tendency following more fusions involved. Nevertheless, the radial distribution of the A/B compartment is not affected dramatically. By contrast, natural populations harboring Rb fusions display significant alterations of chromosome radial location. Overall, K-matrix-originated models enable visualization of chromosomal reorganization with high resolution.

The introduction of single-atom catalysts (SACs) into Fenton-like oxidation promises ultrafast water pollutant elimination, but the limited access to pollutants and oxidant by surface catalytic sites and the intensive oxidant consumption still severely restrict the decontamination performance. While nanoconfinement of SACs allows drastically enhanced decontamination reaction kinetics, the detailed regulatory mechanisms remain elusive. Here, we unveil that, apart from local enrichment of reactants, the catalytic pathway shift is also an important cause for the reactivity enhancement of nanoconfined SACs. The surface electronic structure of cobalt site is altered by confining it within the nanopores of mesostructured silica particles, which triggers a fundamental transition from singlet oxygen to electron transfer pathway for 4-chlorophenol oxidation. The changed pathway and accelerated interfacial mass transfer render the nanoconfined system up to 34.7-fold higher pollutant degradation rate and drastically raised peroxymonosulfate utilization efficiency (from 61.8% to 96.6%) relative to the unconfined control. It also demonstrates superior reactivity for the degradation of other electron-rich phenolic compounds, good environment robustness, and high stability for treating real lake water. Our findings deepen the knowledge of nanoconfined catalysis and may inspire innovations in low-carbon water purification technologies and other heterogeneous catalytic applications. Nanoconfining single metal atom catalysts leads to faster decontamination, primarily due to improved interfacial mass transfer. This study identifies a change in the catalytic pathway as an additional significant factor contributing to the enhanced performance.

Plant movements for survival are nontrivial. Antheridia in the moss Physcomitrium patens (P. patens) use motion to eject sperm in the presence of water. However, the biological and mechanical mechanisms that actuate the process are unknown. Here, the burst of the antheridium of P. patens, triggered by water, results from elastic instability and is determined by an asymmetric change in cell geometry. The tension generated in jacket cell walls of antheridium arises from turgor pressure, and is further promoted when the inner walls of apex burst in hydration, causing water and cellular contents of apex quickly influx into sperm chamber. The outer walls of the jacket cells are strengthened by NAC transcription factor VNS4 and serve as key morphomechanical innovations to store hydrostatic energy in a confined space in P. patens. However, the antheridium in liverwort Marchantia polymorpha (M. polymorpha) adopts a different strategy for sperm release; like jacket cell outer walls of P. patens, the cells surrounding the antheridium of M. polymorpha appear to play a similar role in the storage of energy. Collectively, the work shows that plants have evolved different ingenious devices for sperm discharge and that morphological innovations can differ.

The growing variety of RNA classes, such as mRNAs, lncRNAs, and circRNAs, plays pivotal roles in both developmental processes and various pathophysiological conditions. Nonetheless, our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels. In this study, we combined the CRISPR-RfxCas13d system with sperm-like stem cell-mediated semi-cloning techniques, which enabled the suppressed expression of different RNA species. This approach was employed to interfere with the expression of three types of RNA molecules: Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2(2,3,4,5,6). The results confirmed the critical roles of these RNAs in embryonic development, as their loss led to observable phenotypes, including embryonic lethality, delayed embryonic development, and embryo resorption. In summary, our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.

Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with an X-linked testis-expressed 11 (TEX11) mutation (Tex11PM/Y) identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation. Tex11PM/Y SSCs could be isolated and expanded in vitro normally, and the mutation was corrected by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated endonuclease 9 (Cas9), leading to the generation of repaired SSC lines. Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11PM/Y SSCs, and no predicted off-target sites are modified. Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency. In summary, our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy.

During embryo development, DNA methylation is established by DNMT3A/3B and subsequently maintained by DNMT1. While much research has been done in this field, the functional significance of DNA methylation in embryogenesis remains unknown. Here, we establish a system of simultaneous inactivation of multiple endogenous genes in zygotes through screening for base editors that can efficiently introduce a stop codon. Embryos with mutations in Dnmts and/or Tets can be generated in one step with IMGZ. Dnmt -null embryos display gastrulation failure at E7.5. Interestingly, although DNA methylation is absent, gastrulation-related pathways are down-regulated in Dnmt -null embryos. Moreover, DNMT1, DNMT3A, and DNMT3B are critical for gastrulation, and their functions are independent of TET proteins. Hypermethylation can be sustained by either DNMT1 or DNMT3A/3B at some promoters, which are related to the suppression of miRNAs. The introduction of a single mutant allele of six miRNAs and paternal IG -DMR partially restores primitive streak elongation in Dnmt -null embryos. Thus, our results unveil an epigenetic correlation between promoter methylation and suppression of miRNA expression for gastrulation and demonstrate that IMGZ can accelerate deciphering the functions of multiple genes in vivo.

Mutations in GJB2 (Gap junction protein beta 2) are the most common genetic cause of non-syndromic hereditary deafness in humans, especially the 35delG and 235delC mutations. Owing to the homozygous lethality of Gjb2 mutations in mice, there are currently no perfect mouse models carrying Gjb2 mutations derived from patients for mimicking human hereditary deafness and for unveiling the pathogenesis of the disease. Here, we successfully constructed heterozygous Gjb2+/35delG and Gjb2+/235delC mutant mice through advanced androgenic haploid embryonic stem cell (AG-haESC)-mediated semi-cloning technology, and these mice showed normal hearing at postnatal day (P) 28. A homozygous mutant mouse model, Gjb235delG/35delG, was then generated using enhanced tetraploid embryo complementation, demonstrating that GJB2 plays an indispensable role in mouse placenta development. These mice exhibited profound hearing loss similar to human patients at P14, i.e., soon after the onset of hearing. Mechanistic analyses showed that Gjb2 35delG disrupts the function and formation of intercellular gap junction channels of the cochlea rather than affecting the survival and function of hair cells. Collectively, our study provides ideal mouse models for understanding the pathogenic mechanism of DFNB1A-related hereditary deafness and opens up a new avenue for investigating the treatment of this disease.

Dear Editor, Robertsonian(Rb)fusions contribute to the diversity in species karyotypes.However,how it occurs and propels evolution is still unknown,mainly due to the lack of a simple and efficient method for genetically manipulating chromosomes in animals.While the typical mouse karyotype(2n=40)is completely telocentric except chromo-some Y(ChrY),the western European subspecies M.m.domesticus contains diverse chromosomal races,with a diploid number varying between 38 and 22 due to carrying one or multiple pairs of metacentric chromosomes,which results from the Rb fusions between two telocentric chromosomes.1 Recapitulation of this process in the laboratory may help to shed light on the function of Rb fusions in evolution.The detailed mechanisms underlying Rb fusion in M.m.domesticus requires further investigations,2 but previous studies have shown that Rb fusion-induced metacentric chromosomes have a neo-centromeric region of minor satellite(MinSat)sandwiched between two large blocks of major satellite(MajSat)DNA.3 These observations imply that Rb fusion breakpoints are localized within the MinSat sequences of the centromeric region.Given that CRISPR-Cas9-mediated chromosome engineering has successfully fused 16 yeast chromosomes through head-to-tail strategies to produce yeast strains with 1 or 2 chromosomes,4,5 we hypothesize that CRISPR-Cas9-mediated chromosome engineering through targeting MinSat sequences could be an ideal strategy for creating mouse strains with reduced chromosome numbers in the laboratory.

With the increasing conflict between water availability and demand on a global scale, regulating water user behavior from the bottom-up perspective has gained attention from both water managers and researchers. This paper presents an agent-based model (ABM) to simulate the farmer behavior under changing physical and institutional environments in a case study of Miyun Reservoir watershed, China. The ABM model simulates the complex adaptive systems of agricultural water users under an agricultural water-saving compensation policy. The model we developed in this study characterizes agents' sensitivity, learning capability, and information radius, which impacts agricultural income and water consumption of a household. By applying this model to a local ‘paddy to dryland’ subsidy policy program aiming at reshaping the agricultural water-saving behavior, the results show that this policy program reduces agricultural water consumption while also reducing household agricultural income. The policy depends on administrative intervention and financial subsidies at first to ensure compliance and gradually shape household behavior in the long run. And we also propose a suitable subsidy range of 250–350 yuan for this policy program in the long run. The model we built can help to understand agricultural water use adaptation to both anthropogenic and environmental interventions. This research also contributes to the coupled natural and human systems study with the bottom-up agent-based model, and the empirical case study provides solid policy implications for the agricultural water use allocation and adaptative management at the watershed scale.

Genetic disorders pose as a significant threat to the well-being of human life in this century, as more and more diseases identified are related to mutations presented in patients’ genome [ ]there are limited target sites due to requirement of PAM sequence for DNA cleavage, such as NGG for Cas9 [ ]the safety and ethical issues of the CRISPR applications need re-evaluations along with the discovery of more details about its capabilities [ ]occurring stem cells in adult human tissues are another kinds of stem cells that only have restricted potency (Blau and Daley 2019)

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2, and its RING domain, a histone writer, can ubiquitylate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitylate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.

“人造精子”,也即单倍体胚胎干细胞,为正向遗传学和反向遗传学的研究提供了巨大的帮助,使得研究对象从细胞层面,扩展到更为可信的动物层面.作为胚胎干细胞的一种,“人造精子”拥有着胚胎干细胞可以无限扩增,且保持着很高的多能性的优势;同时,它具备精子的特性,携带有稳定的单倍体基因组遗传信息,能够与卵细胞融合为合子,并发育成健康的半克隆个体. “人造精子”的技术不仅推动了基础研究的速度,而且对医学遗传病的研究和治疗提供了革命性的工具.文章主要讲述了“人造精子”的起源、半克隆技术的实现,以及“人造精子”目前最新的应用研究.

The Human Genome Project (HGP), launched in 1990 and finished in 2004, has not only provided the complete human genome sequence of more than 2.85 billion nucleotides and evidence of 20 000–25 000 protein-coding genes [1], but has also been making huge impacts on biomedical research. One major task of the post-genomic era is to develop the definitive catalog of proteincoding genes, and illustrate proteins’ in vivo dynamic localization and physical interaction. In the past decade, with the rapid development of bioinformatics methodologies, increasingly accurate catalog of protein-coding genes is emerging, with the most recent release containing a total of 21 232 [2]. However, the description of protein properties in vivo is still a challenge, especially at a genome-wide scale, mainly due to the difficulties in systematically producing reliable antibodies for the specific recognition of individual proteins. A generic solution to this problem is to genetically label the protein of interest with a tag, including fluorescent tags for in vivo visualization and affinity tags for the identification of interactions. Due to the advantages of efficient homologous recombination in yeast, protein tagging has been very successful and nearly all protein-coding genes have been tagged at their endogenous genomic loci, enabling global analysis of protein expression, localization and complexes using standard tag-based assays in yeast [3–5]. Genome-scale tagging has also been employed in Caenorhabditis elegans to generate a platform containing tagged worms covering about 73% of the proteome using a system called bacterial artificial chromosome (BAC) TransgeneOmics, in which the tag-coding sequence is inserted into a fosmid as transgenes that include all the important coding and regulatory sequences of a gene [6]. Similarly, a fly genomewide fosmid library of 10 000 green fluorescent protein-tagged clones was generated recently and used to produce a total of 880 transgenic lines for protein localization analysis [7]. In mammals, BAC TransgeneOmics has also been successfully used in human tissue culture cells and mouse embryonic stem cells (ESCs) [8]. Nevertheless, it is extremely difficult to use the high-throughput approach to label protein-coding genes in mammals at the organismal level, such as in the mouse, which is the favored experimental mammal for biomedical studies, restricting large-scale protein analysis just to the cellular level. Moreover, the BAC transgene-based tag strategy may not recapitulate the physiological expression of some proteins due to the random insertion of the transgenes. One potential strategy to endogenously tag every protein inmice is to introduce a tag-coding sequence into the protein-coding gene in ESCs through conventional gene-targeting procedures, followed by the generation of tagged mice via the injection of tagged ESCs into blastocysts for chimera construction and germline transmission. However, chimera formation and germline transmission are always the rate-determining steps of this strategy, thus greatly impeding its large-scale application. Therefore, construction of a genomewide tag-knock-in mouse library is still an unmet need and seems a formidable challenge for the biological research community. Recently, the emergence of two state-of-the-art technologies, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based genome editing and the generation of gene-modified semi-cloned (SC) mice from androgenetic haploid ESCs (AG-haESCs), has made the genomewide tagging of protein-coding genes in mice an achievable scientific objective. CRISPR-Cas9 technology, which originated from the bacterial adaptive immunity system, can rapidly edit a genome with high efficiency and specificity through Cas9-mediated DNA cleavage at specific sites guided by single-guide RNAs (sgRNAs), resulting in DNA modifications by endogenous DNA repair systems [9]. With exogenously supplied DNA, CRISPR-Cas9 can induce precise gene editing at the targeted site, thus enabling efficient in-frame insertions of a tag-coding sequence. AG-haESCs are derived from haploid blastocysts with only the paternal genome [10]; after genetic removal of H19-DMR (differentially DNA methylated region) and IG-DMR, these

In Fig. 2a of this Technical Report originally published, the authors inadvertently used the same set of images for the 4B2N1 and 4B2N3 cells when preparing the figure. The three images (bright field, Oct4-EGFP and pCAG-mRFP) of 4B2N3 cells have now been replaced with the correct versions. The source data for the four cell lines in Fig. 2a, captured in the three independent experiments, have been deposited to Figshare (https://doi.org/10.6084/m9.figshare.7387607.v1), and the figure legends and Methods section have been amended to reflect this. Additionally, the unprocessed blots in Supplementary Fig. 7 corresponding to the top right 'WCL IB: Flag' panel of Fig. 7e were mistakenly duplicates of the unprocessed blots for the bottom left 'IP Flag IB: HA' panel of Fig. 7e, and all unprocessed blots for Supplementary Fig. 6 were mislabelled as blots corresponding to Supplementary Fig. 7. Supplementary Fig. 7 has now been updated to show the correct unprocessed blots for the bottom left 'IP Flag IB: HA' panel of Fig. 7e and to correct the labelling of the unprocessed blots corresponding to Supplementary Fig. 6.

Mammalian haploid embryonic stem cells (haESCs) serve as a powerful tool for genetic analyses at both the cellular and organismal levels. However, spontaneous diploidization of haESCs limits their use in these analyses. Addition of small molecules to the culture medium to control the cell cycle can slow down diploidization, but cell-sorting methods such as FACS are still required to enrich haploid cells for long-term maintenance in vitro. Here, acting on our observation that haploid and diploidized cells differ in diameter, we developed a simplified filtration method to enrich haploid cells from cultured haESCs. We found that regular cell filtration with this system reliably maintained the haploidy of mouse haESCs for over 30 passages. Importantly, CRISPR/Cas9-mediated knockout and knockin were successfully achieved in the filtered cells, leading to stable haploid cell lines carrying the desired gene modifications. Of note, by injecting haESCs into metaphase II oocytes, we efficiently obtained live mice with the expected genetic traits, indicating that regular filtration maintained the functional integrity of haESCs. Moreover, this filtration system was also feasible for derivation of mouse haESCs from parthenogenetic haploid blastocysts and for human haESC maintenance. In conclusion, we have identified a reliable, efficient, and easy-to-handle technique for countering diploidization of haploid cells, a major obstacle in haESC applications.