Interspecies blastocyst complementation holds great potential to address the global shortage of transplantable organs by growing human organs in animals. However, a major challenge in this approach is the limited chimerism of human cells in evolutionarily distant animal hosts due to various xenogeneic barriers. Here, we reveal that human pluripotent stem cells (PSCs) struggle to adhere to animal PSCs. To overcome this barrier, we developed a synthetic biology strategy that leverages nanobody-antigen interactions to enhance interspecies cell adhesion. We engineered cells to express nanobodies and their corresponding antigens on their outer membranes, significantly improving adhesion between different species' PSCs during in vitro assays and increasing the chimerism of human PSCs in mouse embryos. Studying and manipulating interspecies pluripotent cell adhesion will provide valuable insights into cell interaction dynamics during chimera formation and early embryogenesis.

Glycans in corn silk could interact with co-existing small molecules during its absorption, digestion, and biological process. In order to understand the exact mechanism of action of zeaxanthin, it is critical to investigate the biomolecular interactions, which were necessary to form a glycan-small molecule complex and yet produce the bioactive effect. So far, the in-depth study of these natural interactions has not been fully elucidated. Here, we probed that the molecular interaction between zeaxanthin (ZEA) and glycans from corn silk (CSGs) was driven by enthalpy. More importantly, it was the first time found that CSGs can bind to lipid-soluble ZEA could be binded with CSGs. It was the first report on the thermostability of insulin structure and natural glycans. This study should facilitate our understanding of the interaction between lipid soluble molecules and glycans, and provide a more comprehensive understanding of the nutrient base in food.

As the two most principal active substances in the corn silk, polysaccharides and flavonoids, the mechanism of interaction between them has been a topic of intense research. This study provides an in-depth investigation of the interaction mechanism between corn silk glycans and luteoloside (LUT) and the synergistic role that result from this interaction. The interaction mechanism was evaluated by isothermal titration calorimetry (ITC) and circular dichroism (CD), and the synergistic role was evaluated by the expression of glucose transporters (GLUT-1), insulin secretion and surface plasmon resonance (SPR). CD and ITC results indicated that the interaction between CSGs and LUT mainly driven by the Cotton effects, enthalpy and entropy-driven. This interaction precipitated the formation of complexes (CSGs/LUT complexes) between corn silk glycans (CSGs) with four different molecular weights and luteoloside (LUT). Furthermore, the CSGs and LUT play a synergistic role in glucose regulation through GLUT-1 expression and insulin secretion experiments, compared to single luteoloside group.

Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.

AbstractDictyophora rubrovalvata is a valuable fungus homologous to food and medicine, and its polysaccharide have been gaining increasing attention because of its plentiful activity. However, the structure and activity of its homogeneous polysaccharide have not been studied enough. In this study, two polysaccharides DRP-I and DRP-II were purified from D. rubrovalvata. Their structures were characterized by chemical composition, monosaccharide composition analysis, methylation analysis and nuclear magnetic resonance spectroscopy. The results showed that DRP-I and DRP-II were neutral heteropolysaccharides with molecular weights of 5.79 × 103 and 1.25 × 104 Da, respectively, which were composed of mannose, galactose, glucose, xylose and fucose. The main chains were → 6)-α-D-Galp-(1 → 6)-α-D-Galp-(2,1 → 6)-α-D-Manp-(2,1 → 6)-α-D-Galp-(1, and branch chains were β-D-Xylp-(1 → 3)-α-L-Fucp-(1 → 4)-α-D-Manp-(1 → and α-D-Galp-(1 → 3)-α-D-Galp-(1 → . The in vitro immunoactivity assays on dendritic cells showed that DRP-I and DRP-II could up-regulate the expression of IL-10 and IL-6 and inhibit the expression of TNF-α in a concentration-dependent manner. This research indicated that DRP-I and DRP-II possessed immunoactivity by balancing the excessive inflammation, and molecular weight is an important factor affecting immunoactivity. Graphical Abstract

Among the diversified glycan modifications, acylation is one of the most abundant. This modification could be responsible for many of the properties of glycans, such as structural stability and specificity for biological activity. To obtain better insight into the effects of acetylation of glycans on the structure and thermostability of insulin, it is critical to investigate glycans with a high degree of acetylation. An in-depth study of three functional glycans named acetyl-mannan from Dendrobium devonianum (DDAM) was conducted herein by efficient enzymatic depolymerization, and the effect of glycosidic bonds on acetylation modification sites was studied through a molecular dynamics (MD) method, as well as its positive effect on insulin secretion, glucose uptake, and the thermal stability of tertiary structures in vitro. Further study indicated that DDAMs play a hypoglycemic role by sparking the thermostability of the insulin conformation. The hypoglycemic activity displayed a positive correlation with the degree of acetylation in DDAMs. In this work, through the MD method, we confirmed the structure characteristics of DDAMs and provided accurate data support for the structure-activity relationship analysis. Thus, these findings demonstrated that DDAMs might be an exceptional leading compound for the stability of insulin drug.

CRISPR–Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications. Several engineered Cas13 variants achieve targeted RNA degradation with minimal collateral effects.

Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.

RNA knockdown in vivo carries significant potential for disease modelings and therapies. Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes, strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of the genome DNA is not appropriate, and RNA virus infection diseases when pathogenic DNA is not available (such as SARS-Cov-2 and MERS infections). Recently, Cas13d, a family of RNA-targeting CRISPR effectors, has been shown to accomplish robust down-regulation of cellular RNAs in mammalian cells in vitro . Among the various Cas13d subtypes, CasRx (RfxCas13d) showed the most potent RNA knockdown efficiency in HEK293T cells. However, the RNA-targeting activity of Cas13d still needs to be verified in vivo . In this study, the CasRx system was demonstrated to efficiently and functionally knock down genes related to metabolism functions, including Pten, Pcsk9 and lncLstr , in mouse hepatocytes. CasRx-mediated simultaneous knockdown of multiple genes was also achieved by sgRNA arrays, providing a useful strategy to modulate complex metabolism networks. Moreover, the AAV (adeno-associated virus)-mediated delivery of CasRx and Pcsk9 sgRNAs into mouse liver successfully decreased serum PCSK9, resulting in significant reduction of serum cholesterol levels. Importantly, CasRx-mediated knockdown of Pcsk9 is reversible and Pcsk9 could be repeatedly down-regulated, providing an effective strategy to reversibly modulate metabolic genes. The present work supplies a successful proof-of-concept trial that suggests efficient and regulatory knockdown of target metabolic genes for a designed metabolism modulation in the liver.