Corneal alkali burns (CAB) are a severe form of ocular injury that often leads to significant vision loss, with limited effective treatment options available beyond corneal transplantation. Immunity and matrix-regulatory cells (IMRCs) have emerged as a promising alternative due to their ability to modulate immune responses and support tissue repair. This study investigates the efficacy of IMRCs on collagen scaffolds (IMRCs-col) for treating CAB in a rat model. We developed a novel treatment combining IMRCs with a collagen scaffold to align with the ocular surface structure. In vitro analyses showed that IMRCs-col significantly upregulated the expression of immune regulatory molecules, including IL-1RA and SCF. Additionally, IMRCs-col effectively inhibited the production of pro-inflammatory cytokines (IL-8 and Gro-a/CXCL1) while promoting pro-regenerative cytokines (bFGF, HGF, and PDGF). In an animal model of CAB, IMRCs-col transplantation demonstrated substantial efficacy in restoring corneal opacity and reducing neovascularization. Histological examination revealed reduced inflammation and improved corneal tissue regeneration compared to untreated CAB. Enhanced activation of pathways associated with anti-inflammatory responses and tissue repair was observed at days 3, 7, and 21 post-treatment.

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.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Natural cell derivates, including cell sheets (CSs) and matrix gels, have opened new opportunities to probe questions in tissue engineering and regenerative medicine. However, the potential of CSs and hydrogels generated by current protocols is still limited by the challenges of heterogeneity and weak mechanical properties. Here, we developed a 21 day long-term serum-free culture system for human embryonic stem cell (hESC)-derived immunity-and-matrix-regulatory cells (IMRCs). The CSs formed with IMRCs (IMRC-CSs) have a much greater secretion capacity for the extracellular matrix (ECM) and stronger mechanical properties than umbilical cord-derived MSCs, with a ten thousand-fold increase in elastin, a higher elastic modulus of 1500 kPa, a thicker structure of 20.59 μm, and a higher fiber count per square millimeter. The IMRC-CSs could promote corneal chemical injury repair and could be turned into injectable temperature-sensitive hydrogels for uterine adhesion repair via a decellularization process. In summary, we have established a high-strength CS platform using human pluripotent stem cells for the first time, providing a facile and scalable engineering approach for regenerative medicine.

Mesenchymal stromal cells (MSCs) derived from human embryonic stem cells (hESCs) are a desirable cell source for cell therapy owing to their capacity to be produced stably and homogeneously in large quantities. However, a scalable culture system for hPSC-derived MSCs is urgently needed to meet the cell quantity and quality requirements of practical clinical applications. In this study, we developed a new microcarrier with hyaluronic acid (HA) as the core material, which allowed scalable serum-free suspension culture of hESC-derived MSCs (IMRCs). We used optimal microcarriers with a coating collagen concentration of 100 ​μg/mL or concave-structured surface (cHAMCs) for IMRC amplification in a stirred bioreactor, expanding IMRCs within six days with the highest yield of over one million cells per milliliter. In addition, the harvested cells exhibited high viability, immunomodulatory and regenerative therapeutic promise comparable to monolayer cultured MSCs while showing more increased secretion of extracellular matrix (ECM), particularly collagen-related proteins. In summary, we have established a scalable culture system for hESC-MSCs, providing novel approaches for future cell therapies.

In recent years, great strides have been made toward the development of immune cell-based therapies in the treatment of refractory malignancies. Primary T cells and NK cells armed with chimeric antigen receptors have achieved tremendous clinical success especially in patients with leukaemia and lymphoma. However, the autologous origin of these effector cells means that a single batch of laboriously engineered cells treats only a certain patient, leading to high cost, ununiform product quality, and risk of delay in treatment, and therefore results in restricted accessibility of these therapies to the overwhelming majority of the patients. Addressing these tricky obstacles calls for the development of universal immune cell products that can be provided 'off the shelf' in a large amount. Pluripotent stem cells (PSCs), owing to their unique capacity of self-renewal and the potential of multi-lineage differentiation, offer an unlimited cell source to generate uniform and scalable engineered immune cells. This review discusses the major advances in the development of PSC-derived immune cell differentiation approaches and their therapeutic potential in treating both hematologic malignancies and solid tumours. We also consider the potency of PSC-derived immune cells as an alternative therapeutic strategy for other diseases, such as autoimmune diseases, fibrosis, infections, et al.

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.

With the development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas91-based gene editing technology, obtaining gene-knockout cells or animal models has become increasingly convenient. However, other genomic DNA manipulations, including targeted transgene integration or replacement, remain challenging, especially for the treatment of diseases with gene therapy. Targeted transgenes knock-in are achieved using strategies based on intrinsic DNA repair mechanisms such as homologous-directed recombination (HDR)2 and non-homologous end joining (NHEJ).3 Marked efforts have been made to improve the efficacies of these knock-in strategies. Homology-mediated end joining (HMEJ)4 is reported to increase the efficiency of targeted integration by adding a pair of CRISPR/Cas9 targeting sequences flanking the homologous arms of the HDR donor. Homology-independent targeted integration (HITI)5 elevates the frequencies of intended transgene insertion (but not reverse integration) by elaborate arrangement of the orientations of the protospacers in the NHEJ donor. Another technical route to enhance the efficiency of targeted transgene integration is to recruit donor vectors to the targeted genomic site. Some proteins fused to Cas9 can enrich the specific modified single-stranded oligodeoxynucleotide (ssODN) at the double-stranded break (DSB) site to increase the targeted integration efficiency.6-8 However, such systems with chemically modified donor might be unsuitable for delivery in the form of clinically approved viruses such as recombinant adeno-associated viruses (rAAVs), which drastically restrain the application of these vectors in gene therapies. Here, we present a strategy for enhanced HMEJ (enHMEJ) and enhanced HITI (enHITI) by fusing Cas9 with a specific DNA-binding protein, integrase p32, from the human immunodeficiency virus type 1 (HIV-1), to recruit donors harbouring long terminal repeats (LTRs) to the DSB site to increase the targeted integration efficiency in vivo. Following transfection of the plasmid compounds pX330-p32-Cas9-CMV-mRuby2, pPNT6-LTR-GOI and pUC19-U6-sgRNA into cells, the conjugated protein p32-Cas9 is produced from the vector pX330-p32-Cas9-CMV-mRuby2 while single guide RNA (sgRNA) is produced from the vector pUC19-U6-sgRNA. The LTR-flanked transgene is excised from the donor vector by the functional p32-Cas9/sgRNA complex and is then recruited to the genomic cleavage site mediated by the interaction between LTR and the p32 DNA-binding domain (Figure 1A and S1). Initially, we determined whether the p32-Cas9 hybrid protein could bind the LTR in cells. The results of ChIP-qPCR show that there was a strong interaction between p32-Cas9 and LTR (Figure S2). Then to evaluate the contribution of p32/LTRs to the efficiency of HMEJ-based targeted integration, a 293T-enhanced green fluorescent inhibited protein (EGIP) cell line was constructed with a 13-bp deletion in the EGFP coding region driven by a single allelic CAG promoter at the AAVS1 locus (Figure S3). When the deletion is correctly repaired, the 293T-EGIP cells will express functional EGFP. Thus, the absolute efficiency of gene editing could be assessed by fluorescence-activated cell sorting (FACS) (Figure 1B). We found that the knock-in efficiency of enHMEJ (p32/LTR) was 8.61% ± 0.29%, which was significantly higher than the knock-in efficiency of canonical HMEJ (5.19% ± 0.06%) and HDR (2.16% ± 0.08%). These results indicated that there was a 1.5-fold and fourfold increase in targeted integration efficiency for enHMEJ (p32/LTR) compared with HMEJ and HDR respectively (Figure 1C,D). Genotyping analysis of specific missed fragments and sequencing indicated precise gene editing (Figure 1E,F). For in vivo gene integration, any unintended gene expression introduced in the cis-element could be dangerous. The primary sequence of the LTRs we used exhibited promoter activity in 293T cells (Figure 1G). To eliminate this risk, the key 21-bp-terminal region of the LTR, which is responsible for binding to integrase, was retained by removing the U3 enhancer and R promoter region.9 The resulting engineered 21-mer LTR (21 m) did not exhibit promoter activity (Figure 1G). On the other hand, the HIV integrase p32 contains an N-terminal domain (NTD), catalytic core domain (CCD) and C-terminal domain (CTD). The NTD domain is the Zn+ binding domain, which is relative to 3′-processing and strand transfer reactions. The CCD domain was reported to contain a specific DNA-binding site, and the CTD domain contains a non-specific DNA-binding site.9 To address safety concerns, we removed the NTD domain and reassembled the CCD and CTD domains to obtain recombinant protein p32 variant 1 (p32v1) and variant 2 (p32v2; Figure 1H). To investigate whether these engineered elements could increase the knock-in efficiency, the construct combinations were transfected into the 293T-EGIP cell line. Notably, p32v2-Cas9 exhibited the highest knock-in efficiency with the 21 m-modified donor (Figure 1I and S4A). As p32 functions as a dimer, we conjectured that further duplication of p32v2 would enhance the knock-in efficiency, and congruent with this hypothesis, the engineered p32v2 plus (p32v2p) significantly increased the knock-in efficiency from 12.13% ± 0.34% to 15.6% ± 0.7% (Figure 1J and S4B). Compared with the canonical HMEJ, the enHMEJ (p32v2p/21 m) system exhibited a threefold increase in gene knock-in efficiency. To verify whether the optimized system could increase the efficiency of site-specific gene integration at endogenous sites, an IRES-EGFP reporter gene was constructed to target the 3′ untranslated region (UTR) of ACTB and GAPDH in 293T cells and Actb in mouse Hepa1-6 cells (Figure 1K and S5A). Also, a T2A-GFP reporter gene was integrated with the final codon of Sox2 and Nanog in mouse embryonic stem cells (Figure S6A). Seven days after the transfection, enHMEJ (p32v2p/21 m) exhibited the same twofold to threefold increase in knock-in efficiency compared with HMEJ (Figure 1L, S5B and S6B,C). PCR amplification for the integration at the 5′ and 3′ junctions of these targeting sites revealed that the integration based on enHMEJ (p32v2p/21 m) was accurate (Figures S5C,D and S6D,E). HITI is another site-specific and homologous arm-independent targeted integration method. We therefore tested whether our p32v2p/21 m system could enhance HITI efficiency in targeted integration. The 21 m-modified IRES-EGFP gene reporter donor was constructed for ACTB and GAPDH in 293T cells, and Actb in mouse Hepa1-6 cells (Figure 1M and S7A). Seven days after transfection, p32v2p increased the targeted integration efficiency based on HITI by twofold to threefold (Figure 1N and S7B). Genotyping and sequencing analyses showed correct integration of exogenous gene fragments (Figure S7C,D). In conclusion, we developed a simple system to increase the efficiency of gene integration without the need for any ex vivo chemical modification. Importantly, we simplified and optimized our system to erase the potential off-target risks caused by the promoter activity of the LTR and catalytic activity of the integrase p32. Gene-targeted cell lines and animal models will be generated more efficiently with the system. Moreover, the high-efficiency integration in vivo indicates a greater curative effect and lower virus injection dose for gene therapy, which is significant for patients so that they receive a safe treatment and regain their health.10 Thus, the system has considerable potential for gene therapy applications. We appreciate the discussion of all members in Dr Zhou’s lab and Dr Li’s lab. We thank Chao Liu, Cheng Huang, Sinan Ma and Tianshi Pan for their assistance in the experiment. We also thank Qing Meng for the assistance of FACS. This work was supported by the National Key Research and Development Program (2017YFA0103803, 2019YFA0110800 and 2018YFA0107703 to W.L., 2019YFA0903800), National Natural Science Foundation of China (31621004 to W.L.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030400 to W.L.) and CAS Project for Young Scientists in Basic Research (YSBR-012 to W.L.). The authors declare no competing interests. W.L. and Y.Z. conceived this project and supervised the experiments. G.J., C.W., Y.C. and M.D. performed the experiments. G.J. analysed the data. G.J., C.W., Y.C. and Y.Z. wrote the paper with the help from all authors. The data and study materials that support the findings of this study will be available to other researchers from the corresponding authors on reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In this study, we generated an Rbm14 knockout mouse model to explore its functions during early mouse embryogenesis.The Rbm14 knockout mouse model was generated by a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and microinjection techniques. The developmental defects of the knockout embryos were characterized by histological analyses. The accumulation of DNA damage in mouse embryonic stem cells (ESCs) was detected by γH2AX staining and comet assay. The altered mRNA splicing of DNA damage response (DDR)-related genes was detected by RNA-Seq analysis and confirmed by semi-quantitative PCR. The interaction of RBM14 with alternative splicing-related genes was detected by immunoprecipitation-mass spectra (IP-MS) and confirmed by co-immunoprecipitation (Co-IP).Rbm14 knockout in mice results in apoptosis and cell proliferation defects in early post-implantation epiblast cells, leading to gastrulation disruption and embryonic lethality. FACS and immunostaining demonstrate accumulation of DNA damage in Rbm14 knockout ES cells. We also identified altered splicing of DDR-related genes in the knockout mouse ESCs by RNA-Seq, indicating that RBM14-mediated alternative splicing is required for the maintenance of genome integrity during early mouse embryogenesis.Our work reveals that Rbm14 plays an essential role in the maintenance of genome integrity during early mouse embryonic development by regulating alternative splicing of DDR-related genes.

Following fertilization in mammals, the zygote initiates the developmental program, which has a transient capacity to generate cell types of both embryonic and extraembryonic lineages, which is defined as totipotency （Condic, 2014）. In mice,only zygotes and blastomeres of 2-cell stage embryos are considered totipotent, since they have the ability to develop into a full

N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion-induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.

METTL3 catalyzes the formation of N-6-methyl-adenosine (m(6)A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m(6)A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m(6)A modification in spermatogenesis and reproduction in mammals.

To precisely determine the type and status of cells is an important prerequisite for basic researches and regenerative medicine involving stem cells or differentiated cells. However, the traditional destructive cell status examination methods have many limitations, mainly due to the heterogeneity of cells under the reprogramming or differentiation/trans-differentiation process. Here we present a new method to non-destructively determine the pluripotent level of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), or the types of differentiated cells. The method is achieved by examining the expression profiles of microRNAs (miRNAs) in cell culture medium, which show consistent abundance trend as those of the cellular miRNAs. Therefore, the method enables status examination and afterward application being achieved on the same population of cells, which will greatly facilitate cell reprogramming or differentiation/trans-differentiation related based research and clinical therapy.