Nuclear RNA homeostasis promotes systems-level coordination of cell fate and senescence.

Understanding cellular coordination remains a challenge despite knowledge of individual pathways. The RNA exosome, targeting a wide range of RNA substrates, is often downregulated in cellular senescence. Utilizing an auxin-inducible system, we observed that RNA exosome depletion in embryonic stem cells significantly affects the transcriptome and proteome, causing pluripotency loss and pre-senescence onset. Mechanistically, exosome depletion triggers acute nuclear RNA aggregation, disrupting nuclear RNA-protein equilibrium. This disturbance limits nuclear protein availability and hinders polymerase initiation and engagement, reducing gene transcription. Concurrently, it promptly disrupts nucleolar transcription, ribosomal processes, and nuclear exporting, resulting in a translational shutdown. Prolonged exosome depletion induces nuclear structural changes resembling senescent cells, including aberrant chromatin compaction, chromocenter disassembly, and intensified heterochromatic foci. These effects suggest that the dynamic turnover of nuclear RNA orchestrates crosstalk between essential processes to optimize cellular function. Disruptions in nuclear RNA homeostasis result in systemic functional decline, altering the cell state and promoting senescence.

A knowledge-guided pre-training framework for improving molecular representation learning.

Learning effective molecular feature representation to facilitate molecular property prediction is of great significance for drug discovery. Recently, there has been a surge of interest in pre-training graph neural networks (GNNs) via self-supervised learning techniques to overcome the challenge of data scarcity in molecular property prediction. However, current self-supervised learning-based methods suffer from two main obstacles: the lack of a well-defined self-supervised learning strategy and the limited capacity of GNNs. Here, we propose Knowledge-guided Pre-training of Graph Transformer (KPGT), a self-supervised learning framework to alleviate the aforementioned issues and provide generalizable and robust molecular representations. The KPGT framework integrates a graph transformer specifically designed for molecular graphs and a knowledge-guided pre-training strategy, to fully capture both structural and semantic knowledge of molecules. Through extensive computational tests on 63 datasets, KPGT exhibits superior performance in predicting molecular properties across various domains. Moreover, the practical applicability of KPGT in drug discovery has been validated by identifying potential inhibitors of two antitumor targets: hematopoietic progenitor kinase 1 (HPK1) and fibroblast growth factor receptor 1 (FGFR1). Overall, KPGT can provide a powerful and useful tool for advancing the artificial intelligence (AI)-aided drug discovery process.

A split and inducible adenine base editor for precise in vivo base editing.

DNA base editors use deaminases fused to a programmable DNA-binding protein for targeted nucleotide conversion. However, the most widely used TadA deaminases lack post-translational control in living cells. Here, we present a split adenine base editor (sABE) that utilizes chemically induced dimerization (CID) to control the catalytic activity of the deoxyadenosine deaminase TadA-8e. sABE shows high on-target editing activity comparable to the original ABE with TadA-8e (ABE8e) upon rapamycin induction while maintaining low background activity without induction. Importantly, sABE exhibits a narrower activity window on DNA and higher precision than ABE8e, with an improved single-to-double ratio of adenine editing and reduced genomic and transcriptomic off-target effects. sABE can achieve gene knockout through multiplex splice donor disruption in human cells. Furthermore, when delivered via dual adeno-associated virus vectors, sABE can efficiently convert a single A•T base pair to a G•C base pair on the PCSK9 gene in mouse liver, demonstrating in vivo CID-controlled DNA base editing. Thus, sABE enables precise control of base editing, which will have broad implications for basic research and in vivo therapeutic applications.

Explore the dominant factor in prime editing via a view of DNA processing.

Prime editing is a revolutionary gene-editing method that is capable of introducing insertions, deletions and base substitutions into the genome. However, the editing efficiency of Prime Editor (PE) is limited by the DNA repair process. Here, we show that overexpression of the flap structure-specific endonuclease 1 (FEN1) and the DNA ligase 1 (LIG1) increases the efficiency of prime editing, which is similar to the dominant negative mutL homolog 1 (MLH1dn). In addition, MLH1 is still the dominant factor over FEN1 and LIG1 in prime editing. Our results help to further understand the relationship of proteins involved in prime editing and envisage future directions for the development of PE.

Engineered PROTAC-CID Systems for Mammalian Inducible Gene Regulation.

Gene regulation via chemically induced dimerization (CID) is useful for biomedical research. However, the number, type, versatility, and in vivo applications of CID tools remain limited. Here, we demonstrate the development of proteolysis-targeting chimera-based scalable CID (PROTAC-CID) platforms by systematically engineering the available PROTAC systems for inducible gene regulation and gene editing. Further, we show orthogonal PROTAC-CIDs that can fine-tune gene expression at gradient levels or multiplex biological signals with different logic gating operations. Coupling the PROTAC-CID platform with genetic circuits, we achieve digitally inducible expression of DNA recombinases, base- and prime-editors for transient genome manipulation. Finally, we package a compact PROTAC-CID system into adeno-associated viral vectors for inducible and reversible gene activation in vivo. This work provides a versatile molecular toolbox that expands the scope of chemically inducible gene regulation in human cells and mice.

Sense-and-Respond Payload Delivery Using a Novel Antigen-Inducible Promoter Improves Suboptimal CAR-T Activation.

Chimeric antigen receptor (CAR)-T cell therapies demonstrate the clinical potential of lymphocytes engineered with synthetic properties. However, CAR-T cells are ineffective in most solid tumors, partly due to inadequate activation of the infused lymphocytes at the site of malignancy. To selectively enhance antitumor efficacy without exacerbating off-target toxicities, CAR-T cells can be engineered to preferentially deliver immunostimulatory payloads in tumors. Here, we report a novel antigen-inducible promoter for conditional payload expression in primary human T cells. In therapeutic T cell models, the novel NR4A-based promoter induced higher reporter gene expression than the conventional NFAT-based promoter under weakly immunogenic conditions, where payload expression is most needed. Minimal activity was detected from the inducible promoters in the absence of antigen and after withdrawal of stimulation. As a functional proof-of-concept, we used the NR4A-based promoter to express cytokines in an antimesothelin CAR-T model with suboptimal stimulation and observed improved proliferation compared to T cells engineered with the conventional NFAT promoter or CAR alone. Our system achieves CAR-directed payload expression under weakly immunogenic conditions and could enable the next generation of cell therapies with enhanced antitumor efficacy.

Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity.

The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to measure the relative cleavage rate between off- and on-target sequences (off-on ratios) of 1902 gRNAs on 13,314 synthetic target sequences, and reveal a set of sequence rules involving 2 factors in off-targeting: 1) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context; 2) an "epistasis-like" combinatorial effect of multiple mismatches, which are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. These sequence rules lead to the development of MOFF, a model-based predictor of Cas9-mediated off-target effects. Moreover, the "epistasis-like" combinatorial effect suggests a strategy of allele-specific genome editing using mismatched guides. With the aid of MOFF prediction, this strategy significantly improves the selectivity and expands the application domain of Cas9-based allele-specific editing, as tested in a high-throughput allele-editing screen on 18 cancer hotspot mutations.

Magnetic Living Hydrogels for Intestinal Localization, Retention, and Diagnosis.

Natural microbial sensing circuits can be rewired into new gene networks to build living sensors that detect and respond to disease-associated biomolecules. However, synthetic living sensors, once ingested, are cleared from the gastrointestinal (GI) tract within 48 hours; retaining devices in the intestinal lumen is prone to intestinal blockage or device migration. To localize synthetic microbes and safely extend their residence in the GI tract for health monitoring and sustained drug release, an ingestible magnetic hydrogel carrier is developed to transport diagnostic microbes to specific intestinal sites. The magnetic living hydrogel is localized and retained by attaching a magnet to the abdominal skin, resisting the peristaltic waves in the intestine. The device retention is validated in a human intestinal phantom and an in vivo rodent model, showing that the ingestible hydrogel maintains the integrated living bacteria for up to seven days, which allows the detection of heme for GI bleeding in the harsh environment of the gut. The retention of microelectronics is also demonstrated by incorporating a temperature sensor into the magnetic hydrogel carrier.

Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information.

Although Cas9 nucleases are remarkably diverse in microorganisms, the range of genomic sequences targetable by a CRISPR/Cas9 system is restricted by the requirement of a short protospacer adjacent motif (PAM) at the target site. Here, we generate a group of chimeric Cas9 (cCas9) variants by replacing the key region in the PAM interaction (PI) domain of Staphylococcus aureus Cas9 (SaCas9) with the corresponding region in a panel of SaCas9 orthologs. By using a functional assay at target sites with different nucleotide recombinations at PAM position 3-6, we identify several cCas9 variants with expanded recognition capability at NNVRRN, NNVACT, NNVATG, NNVATT, NNVGCT, NNVGTG, and NNVGTT PAM sequences. In summary, we provide a panel of cCas9 variants accessible up to 1/4 of all the possible genomic targets in mammalian cells.

Network-Based Combinatorial CRISPR-Cas9 Screens Identify Synergistic Modules in Human Cells.

Tumorigenesis is a complex process that is driven by a combination of networks of genes and environmental factors; however, efficient approaches to identifying functional networks that are perturbed by the process of tumorigenesis are lacking. In this study, we provide a comprehensive network-based strategy for the systematic discovery of functional synergistic modules that are causal determinants of inflammation-induced tumorigenesis. Our approach prioritizes candidate genes selected by integrating clinical-based and network-based genome-wide gene prediction methods and identifies functional synergistic modules based on combinatorial CRISPR-Cas9 screening. On the basis of candidate genes inferred de novo from experimental and computational methods to be involved in inflammation and cancer, we used an existing TGFß1-induced cellular transformation model in colonic epithelial cells and a new combinatorial CRISPR-Cas9 screening strategy to construct an inflammation-induced differential genetic interaction network. The inflammation-induced differential genetic interaction network that we generated yielded functional insights into the genes and functional module combinations, and showed varied responses to the inflammation agents as well as active traditional Chinese medicine compounds. We identified opposing differential genetic interactions of inflammation-induced tumorigenesis: synergistic promotion and suppression. The synergistic promotion state was primarily caused by deletions in the immune and metabolism modules; the synergistic suppression state was primarily induced by deletions in the proliferation and immune modules or in the proliferation and metabolism modules. These results provide insight into possible early combinational targets and biomarkers for inflammation-induced tumorigenesis and highlight the synergistic effects that occur among immune, proliferation, and metabolism modules. In conclusion, this approach deepens the understanding of the underlying mechanisms that cause inflammation to potentially increase the cancer risk of colonic epithelial cells and accelerate the translation into novel functional modules or synergistic module combinations that modulate complex disease phenotypes.

Rational Design of Mini-Cas9 for Transcriptional Activation.

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells.

Programmable and precise regulation of dCas9 functions in response to multiple molecular signals by using synthetic gene circuits will expand the application of the CRISPR-Cas technology. However, the application of CRISPR-Cas therapeutic circuits is still challenging due to the restrictive cargo size of existing viral delivery vehicles. Here, we construct logic AND circuits by integrating multiple split dCas9 domains, which is useful to reduce the size of synthetic circuits. In addition, we engineer sensory switches by exchanging split dCas9 domains, allowing differential regulations on one gene, or activating two different genes in response to cell-type specific microRNAs. Therefore, we provide a valuable split-dCas9 toolkit to engineer complex transcription controls, which may inspire new biomedical applications.