All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Asymmetric Expression of LincGET Biases Cell Fate in Two-Cell Mouse Embryos.

In early mammalian embryos, it remains unclear how the first cell fate bias is initially triggered and amplified toward cell fate segregation. Here, we report that a long noncoding RNA, LincGET, is transiently and asymmetrically expressed in the nucleus of two- to four-cell mouse embryos. Overexpression of LincGET in one of the two-cell blastomeres biases its progeny predominantly toward the inner cell mass (ICM) fate. Mechanistically, LincGET physically binds to CARM1 and promotes the nuclear localization of CARM1, which can further increase the level of H3 methylation at Arginine 26 (H3R26me), activate ICM-specific gene expression, upregulate transposons, and increase global chromatin accessibility. Simultaneous overexpression of LincGET and depletion of Carm1 no longer biased embryonic fate, indicating that the effect of LincGET in directing ICM lineage depends on CARM1. Thus, our data identify LincGET as one of the earliest known lineage regulators to bias cell fate in mammalian 2-cell embryos.

Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

Refining computational inference of gene regulatory networks: integrating knockout data within a multi-task framework.

Constructing accurate gene regulatory network s (GRNs), which reflect the dynamic governing process between genes, is critical to understanding the diverse cellular process and unveiling the complexities in biological systems. With the development of computer sciences, computational-based approaches have been applied to the GRNs inference task. However, current methodologies face challenges in effectively utilizing existing topological information and prior knowledge of gene regulatory relationships, hindering the comprehensive understanding and accurate reconstruction of GRNs. In response, we propose a novel graph neural network (GNN)-based Multi-Task Learning framework for GRN reconstruction, namely MTLGRN. Specifically, we first encode the gene promoter sequences and the gene biological features and concatenate the corresponding feature representations. Then, we construct a multi-task learning framework including GRN reconstruction, Gene knockout predict, and Gene expression matrix reconstruction. With joint training, MTLGRN can optimize the gene latent representations by integrating gene knockout information, promoter characteristics, and other biological attributes. Extensive experimental results demonstrate superior performance compared with state-of-the-art baselines on the GRN reconstruction task, efficiently leveraging biological knowledge and comprehensively understanding the gene regulatory relationships. MTLGRN also pioneered attempts to simulate gene knockouts on bulk data by incorporating gene knockout information.

SIRT6 deficiency results in developmental retardation in cynomolgus monkeys.

SIRT6 acts as a longevity protein in rodents1,2. However, its biological function in primates remains largely unknown. Here we generate a SIRT6-null cynomolgus monkey (Macaca fascicularis) model using a CRISPR-Cas9-based approach. SIRT6-deficient monkeys die hours after birth and exhibit severe prenatal developmental retardation. SIRT6 loss delays neuronal differentiation by transcriptionally activating the long non-coding RNA H19 (a developmental repressor), and we were able to recapitulate this process in a human neural progenitor cell differentiation system. SIRT6 deficiency results in histone hyperacetylation at the imprinting control region of H19, CTCF recruitment and upregulation of H19. Our results suggest that SIRT6 is involved in regulating development in non-human primates, and may provide mechanistic insight into human perinatal lethality syndrome.

Live-cell imaging reveals redox metabolic reprogramming during zygotic genome activation.

Metabolic programming is deeply intertwined with early embryonic development including zygotic genome activation (ZGA), the polarization of zygotic cells, and cell fate commitment. It is crucial to establish a noninvasive imaging technology that spatiotemporally illuminates the cellular metabolism pathways in embryos to track developmental metabolism in situ. In this study, we used two high-quality genetically encoded fluorescent biosensors, SoNar for NADH/NAD+ and iNap1 for NADPH, to characterize the dynamic regulation of energy metabolism and redox homeostasis during early zygotic cleavage. Our imaging results showed that NADH/NAD+ levels decreased from the early to the late two-cell stage, whereas the levels of the reducing equivalent NADPH increased. Mechanistically, transcriptome profiling suggested that during the two-cell stage, zygotic cells downregulated the expression of genes involved in glucose uptake and glycolysis, and upregulated the expression of genes for pyruvate metabolism in mitochondria and oxidative phosphorylation, with a decline in the expression of two peroxiredoxin genes, Prdx1 and Prdx2. Collectively, with the establishment of in situ metabolic monitoring technology, our study revealed the programming of redox metabolism during ZGA.

Impaired lipid metabolism by age-dependent DNA methylation alterations accelerates aging.

Epigenetic alterations and metabolic dysfunction are two hallmarks of aging. However, the mechanism of how their interaction regulates aging, particularly in mammals, remains largely unknown. Here we show ELOVL fatty acid elongase 2 (Elovl2), a gene whose epigenetic alterations are most highly correlated with age prediction, contributes to aging by regulating lipid metabolism. Impaired Elovl2 function disturbs lipid synthesis with increased endoplasmic reticulum stress and mitochondrial dysfunction, leading to key accelerated aging phenotypes. Restoration of mitochondrial activity can rescue age-related macular degeneration (AMD) phenotypes induced by Elovl2 deficiency in human retinal pigmental epithelial (RPE) cells. We revealed an epigenetic-metabolism axis contributing to aging and potentially to antiaging therapy.

Long noncoding RNA lnc-NAP sponges mmu-miR-139-5p to modulate Nanog functions in mouse ESCs and embryos.

The pluripotency of embryonic stem cells (ESCs) is controlled by a multilayer regulatory network, of which the key factors include core pluripotency genes Oct4, Sox2 and Nanog, and multiple microRNAs (miRNAs). Recently, long noncoding RNAs (lncRNAs) have been discovered as a class of new regulators for ESCs, and some lncRNAs could function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we identify mmu-miR-139-5p as a new regulator for Nanog by targeting Nanog 3' untranslated region (UTR) to repress Nanog expression in mouse ESCs and embryos. Such regulation could be released by an ESC-specifically expressed ceRNA named lnc-NAP. The expression of lnc-NAP is activated by OCT4, SOX2, as well as NANOG through promoter binding. Downregulation of lnc-NAP reduces Nanog abundance, which leads to decreased pluripotency of mouse ESCs and embryonic lethality. These results reveal lnc-NAP as a new regulator for Nanog in mouse ESCs, and uncover a feed-forward regulatory loop of Nanog through the participation of lnc-NAP.

Rat embryonic stem cells produce fertile offspring through tetraploid complementation.

Pluripotency of embryonic stem cells (ESCs) can be functionally assessed according to the developmental potency. Tetraploid complementation, through which an entire organism is produced from the pluripotent donor cells, is taken as the most stringent test for pluripotency. It remains unclear whether ESCs of other species besides mice can pass this test. Here we show that the rat ESCs derived under 2i (two small molecule inhibitors) conditions at very early passages are able to produce fertile offspring by tetraploid complementation. However, they lose this capacity rapidly during culture due to a nearly complete loss of genomic imprinting. Our findings support that the naïve ground state pluripotency can be captured in rat ESCs but also point to the species-specific differences in its regulation and maintenance, which have implications for the derivation and application of naïve pluripotent stem cells in other species including human.

Mitochondrially produced ATP affects stem cell pluripotency via Actl6a-mediated histone acetylation.

ATP is mainly generated by glycolysis in pluripotent stem cells (PSCs) and is consumed to maintain cell viability. Differences in mitochondrial activity among induced (i)PSCs with different degrees of pluripotency are poorly understood. In this study, by comparing gene expression and mitochondrial activity among iPSCs with different degrees of pluripotency, we found that mitochondrial complex I gene expression, complex I activity, and cellular ATP levels were much higher in fully pluripotent stem cell lines than in partially pluripotent stem cell lines. Actin-like protein 6a (Actl6a), a component of ATP-dependent chromatin remodeling and histone acetylation complexes, was more highly expressed in fully pluripotent stem cell lines. ATP promoted Actl6a expression and histone acetylation. Actl6a knockdown reduced the pluripotency of embryonic stem cells (ESCs), and this reduction could not be rescued by the addition of ATP. Furthermore, inhibiting ATP formation by treatment with rotenone reduced the pluripotency of ESCs. These data suggest that the abundance of mitochondrially produced ATP affects stem cell pluripotency via Actl6a-mediated histone acetylation.-Zhang, Y., Cui, P., Li, Y., Feng, G., Tong, M., Guo, L., Li, T., Liu, L., Li, W., Zhou, Q. Mitochondrially produced ATP affects stem cell pluripotency via Actl6a-mediated histone acetylation.

MicroRNA-494 promotes cancer progression and targets adenomatous polyposis coli in colorectal cancer.

BACKGROUND: Aberrant activation of the Wnt/ß-catenin signaling pathway is frequently observed in colorectal cancer (CRC). ß-catenin is the major Wnt signaling pathway effector and inactivation of adenomatous polyposis coli (APC) results in nuclear accumulation of ß-catenin. It has been suggested that inactivation of APC plays an important role in activation of the Wnt/ß-catenin pathway and in the progression of colorectal tumorigenesis. However, the mechanism through which APC mediates colorectal tumorigenesis is not understood. Increasing evidence suggests that the dysregulation of microRNAs (miRNAs) is involved in colorectal tumorigenesis. Although miR-494 has been reported as being an upregulated miRNA, the interplay between miR-494 and APC-mediated colorectal tumorigenesis progression remains unclear. METHODS: The expression of miR-494 in tissues from patients diagnosed with CRC was analyzed using a microarray and real-time PCR. The effects of miR-494 on cell proliferation and tumorigenesis in CRC cells were analyzed by flow cytometry, colony formation assays, BrdU incorporation assays, and CCK8 assays. The correlation between miR-494 expression and APC expression, as well as the mechanisms by which miR-494 regulates APC in CRC were also addressed. RESULTS: miR-494 was significantly upregulated in CRC tissues, and this increase was negatively associated with APC expression. APC was confirmed to be a direct target of miR-494 in CRC. Furthermore, overexpression of miR-494 induced Wnt/ß-catenin signaling by targeting APC, thus promoting CRC cell growth. CONCLUSIONS: This study provides novel insights into the role of miR-494 in controlling CRC cell proliferation and tumorigenesis, and identifies miR-494 as a potential prognostic marker and therapeutic target.

RBM14 is indispensable for pluripotency maintenance and mesoderm development of mouse embryonic stem cells.

The pluripotency of embryonic stem cells (ESCs) is maintained by core pluripotency transcription factors, cofactors and several signaling pathways. RBM14 is a component of the para-speckle complex, which has been implicated in multiple important biological processes. The role of RBM14 in ESCs and lineage differentiation remains to be elucidated. In the present study, we provided evidence that RBM14 plays important roles in maintaining pluripotency and in the early differentiation of ESCs. RBM14 was demonstrated to be expressed in mouse embryonic stem cells (mESCs) and localized in the nucleus. RBM14 expression was depleted in mESCs using clustered regularly interspaced short palindromic repeats (CRISPR) technology. Our results also showed that RBM14 depletion altered the gene expression profiles of mESCs. In particular, pluripotency-associated genes and genes involved in the Wnt and TGF-ß signaling pathways were downregulated in RBM14 knockout mESCs. Furthermore, RBM14 was found to be essential for mesoderm development in vitro and in vivo. The specific effects of RBM14 depletion were verified by conducting a rescue experiment. Our findings demonstrated that RBM14 not only plays an important role in maintaining the pluripotency of mESCs but is also indispensable for mesoderm development.

Ubiquitously expressed genes participate in cell-specific functions via alternative promoter usage.

How do different cell types acquire their specific identities and functions is a fundamental question of biology. Previously significant efforts have been devoted to search for cell-type-specifically expressed genes, especially transcription factors, yet how do ubiquitously expressed genes participate in the formation or maintenance of cell-type-specific features remains largely unknown. Here, we have identified 110 mouse embryonic stem cell (mESC) specifically expressed transcripts with cell-stage-specific alternative transcription start sites (SATS isoforms) from 104 ubiquitously expressed genes, majority of which have active epigenetic modification- or stem cell-related functions. These SATS isoforms are specifically expressed in mESCs, and tend to be transcriptionally regulated by key pluripotency factors through direct promoter binding. Knocking down the SATS isoforms of Nmnat2 or Usp7 leads to differentiation-related phenotype in mESCs. These results demonstrate that cell-type-specific transcription factors are capable to produce cell-type-specific transcripts with alternative transcription start sites from ubiquitously expressed genes, which confer ubiquitously expressed genes novel functions involved in the establishment or maintenance of cell-type-specific features.

Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis.

Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.

Elimination of GGTA1, CMAH, ß4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions.

BACKGROUND: Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic, while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs. Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection (HAR) that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure, in which process the MHC II molecule plays critical roles. METHODS: Thus, we generate a 4-gene (GGTA1, CMAH, ß4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously. RESULTS: We successfully obtained 4KO piglets with deficiency in all alleles of genes, and at cellular and tissue levels. Additionally, the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping. Piglets have survived for more than one year in the barrier, and also survived for more than 3 months in the conventional environment, suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment. CONCLUSIONS: 4KO piglets have lower immunogenicity, are safe in genomic level, and are easier to breed than the model with both MHC I and II deletion.

Alternative splicing of CARM1 regulated by LincGET-guided paraspeckles biases the first cell fate in mammalian early embryos.

The heterogeneity of CARM1 controls first cell fate bias during early mouse development. However, how this heterogeneity is established is unknown. Here, we show that Carm1 mRNA is of a variety of specific exon-skipping splicing (ESS) isoforms in mouse two-cell to four-cell embryos that contribute to CARM1 heterogeneity. Disruption of paraspeckles promotes the ESS of Carm1 precursor mRNAs (pre-mRNAs). LincGET, but not Neat1, is required for paraspeckle assembly and inhibits the ESS of Carm1 pre-mRNAs in mouse two-cell to four-cell embryos. We further find that LincGET recruits paraspeckles to the Carm1 gene locus through HNRNPU. Interestingly, PCBP1 binds the Carm1 pre-mRNAs and promotes its ESS in the absence of LincGET. Finally, we find that the ESS seen in mouse two-cell to four-cell embryos decreases CARM1 protein levels and leads to trophectoderm fate bias. Our findings demonstrate that alternative splicing of CARM1 has an important role in first cell fate determination.

High-efficiency targeted transgene integration via primed micro-homologues.

Due to the difficulties in precisely manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes triggered by double-strand breaks is inherently inefficient. Here, we exploit prime editors to devise a robust knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT), which utilizes reverse-transcribed single-stranded micro-homologues to boost targeted KIs in different types of cells. The improved version of PAINT, designated PAINT 3.0, maximizes editing efficiency and minimizes off-target integration, especially in dealing with scarless in-frame KIs. Using PAINT 3.0, we target a reporter transgene into housekeeping genes with editing efficiencies up to 80%, more than 10-fold higher than the traditional homology-directed repair method. Moreover, the use of PAINT 3.0 to insert a 2.5-kb transgene achieves up to 85% KI frequency at several therapeutically relevant genomic loci, suggesting its potential for clinical applications. Finally, PAINT 3.0 enables high-efficiency non-viral genome targeting in primary T cells and produces functional CAR-T cells with specific tumor-killing ability. Thus, we establish that the PAINT method is a powerful gene editing tool for large transgene integrations and may open new avenues for cell and gene therapies and genome writing technologies.

Characterisation of X chromosome status of human extended pluripotent stem cells.

Different pluripotent cell types have been established by capturing pluripotency in different states. Human extended pluripotent stem cells (hEPSCs), recently established by two independent studies, have the capability of differentiating into both embryonic and extraembryonic lineages, as well as forming human blastoids, showing great potential for early human development modeling and regenerative medicine. Considering that X chromosome status in female human pluripotent stem cells is dynamic and heterogeneous, and often leads to functional consequences, we characterized it in hEPSCs. We derived hEPSCs from primed human embryonic stem cells (hESCs) with defined X chromosome status (pre- or post-X chromosome inactivation) using two previously published methods. We showed that hEPSCs derived using both methods had highly similar transcription profiles and X chromosome status. However, the X chromosome status of hEPSCs is largely determined by the primed hESCs from which they were derived, suggesting a lack of complete reprogramming of X chromosome during primed to extended/expanded pluripotency conversion. Furthermore, we found that the X chromosome status of hEPSCs affected their ability to differentiate into embryonic or extraembryonic lineage cells. Taken together, our work characterized the X chromosome status of hEPSCs, providing important information for the future application of hEPSCs.

Safety and efficacy of human ESC-derived corneal endothelial cells for corneal endothelial dysfunction.

BACKGROUND: Research on human pluripotent stem cells (hPSCs) has shown tremendous progress in cell-based regenerative medicine. Corneal endothelial dysfunction is associated with the loss and degeneration of corneal endothelial cells (CECs), rendering cell replacement a promising therapeutic strategy. However, comprehensive preclinical assessments of hPSC-derived CECs for this cell therapy remain a challenge. RESULTS: Here we defined an adapted differentiation protocol to generate induced corneal endothelial cells (iCECs) consistently and efficiently from clinical-grade human embryonic stem cells (hESCs) with xeno-free medium and manufactured cryopreserved iCECs. Cells express high levels of typical CECs markers and exhibit transendothelial potential properties in vitro typical of iCECs. After rigorous quality control measures, cells meeting all release criteria were available for in vivo studies. We found that there was no overgrowth or tumorigenicity of grafts in immunodeficient mice. After grafting into rabbit models, the surviving iCECs ameliorated edema and recovered corneal opacity. CONCLUSIONS: Our work provides an efficient approach for generating iCECs and demonstrates the safety and efficacy of iCECs in disease modeling. Therefore, clinical-grade iCECs are a reliable source for future clinical treatment of corneal endothelial dysfunction.

tRF-Gln-CTG-026 ameliorates liver injury by alleviating global protein synthesis.

tsRNAs (tRNA-derived small RNAs), as products of the stress response, exert considerable influence on stress response and injury regulation. However, it remains largely unclear whether tsRNAs can ameliorate liver injury. Here, we demonstrate the roles of tsRNAs in alleviating liver injury by utilizing the loss of NSun2 (NOP2/Sun domain family, member 2) as a tsRNAs-generating model. Mechanistically, the loss of NSun2 reduces methyluridine-U5 (m5U) and cytosine-C5 (m5C) of tRNAs, followed by the production of various tsRNAs, especially Class I tsRNAs (tRF-1s). Through further screening, we show that tRF-Gln-CTG-026 (tG026), the optimal tRF-1, ameliorates liver injury by repressing global protein synthesis through the weakened association between TSR1 (pre-rRNA-processing protein TSR1 homolog) and pre-40S ribosome. This study indicates the potential of tsRNA-reduced global protein synthesis in liver injury and repair, suggesting a potential therapeutic strategy for liver injury.