Natural materials typically exhibit irregular and non-periodic architectures, endowing them with compelling functionalities such as body protection, camouflage, and mechanical stress modulation. Among these functionalities, mechanical stress modulation is crucial for homeostasis regulation and tissue remodeling. Here, we uncover the relationship between stress modulation functionality and the irregularity of bio-inspired architected materials by a generative computational framework. This framework optimizes the spatial distribution of a limited set of basic building blocks and uses these blocks to assemble irregular materials with heterogeneous, disordered microstructures. Despite being irregular and non-periodic, the assembled materials display spatially varying properties that precisely modulate stress distribution towards target values in various control regions and load cases, echoing the robust stress modulation capability of natural materials. The performance of the generated irregular architected materials is experimentally validated with 3D printed physical samples - a good agreement with target stress distribution is observed. Owing to its capability to redirect loads while keeping a proper amount of stress to stimulate bone repair, we demonstrate the potential application of the stress-programmable architected materials as support in orthopedic femur restoration.
Stress, Mechanical , Printing, Three-Dimensional , Humans , Femur , Biomimetic Materials/chemistry , Tissue Engineering/methods , Materials Testing , Tissue Scaffolds/chemistry
Recently, a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize a minimal NG protospacer adjacent motif (PAM) was reported to expand the targeting scope in genome editing. However, increased genome-wide off-target mutations with this variant compared with SpCas9 were reported in previous studies. In addition, lower base editing frequencies and higher unintended off-target mutations were also found in Hoxc13-ablated rabbits generated by NG-BE4max in our study. Here, a high-fidelity base editor, NG-HiFi, in comparison to NG-BE4max, showed retention of on-target activity while exhibiting significantly decreased off-target activity in Hoxc13-ablated rabbits. Collectively, the improved specificity and reduced off-target effect of SpCas9-NG assisted in cytidine base editing with the NG-HiFi system, providing a promising tool to precisely model human diseases in rabbits.
Cytidine base editor (CBE), which is composed of a cytidine deaminase fused to Cas9 nickase, has been widely used to induce C-to-T conversions in a wide range of organisms. However, the targeting scope of current CBEs is largely restricted to protospacer adjacent motif (PAM) sequences containing G, T, or A bases. In this study, we developed a new base editor termed "nNme2-CBE" with excellent PAM compatibility for cytidine dinucleotide, significantly expanding the genome-targeting scope of CBEs. Using nNme2-CBE, targeted editing efficiencies of 29.0%-55.0% and 17.3%-52.5% were generated in human cells and rabbit embryos, respectively. In contrast to conventional nSp-CBE, the nNme2-CBE is a natural high-fidelity base editing platform with minimal DNA off-targeting detected in vivo. Significantly increased efficiency in GC context and precision were determined by combining nNme2Cas9 with rationally engineered cytidine deaminases. In addition, the Founder rabbits with accurate single-base substitutions at Fgf5 gene loci were successfully generated by using the nNme2-CBE system. These novel nNme2-CBEs with expanded PAM compatibility and high fidelity will expand the base editing toolset for efficient gene modification and therapeutic applications.
CRISPR-Associated Protein 9/metabolism , Cytidine Deaminase/metabolism , Cytidine/metabolism , Nucleotide Motifs , Animals , Fluorescence , Humans , RNA Editing , Rabbits
BACKGROUND: Cytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas9 nickase (nCas9), enable efficient C-to-T conversion in various organisms. However, current base editors can induce unwanted bystander C-to-T conversions when multiple Cs are present in the ~ 5-nucleotide activity window of cytidine deaminase, which negatively affects their precision. Here, we develop a new base editor which significantly reduces unwanted bystander activities. RESULTS: We used an engineered human APOBEC3G (eA3G) C-terminal catalytic domain with preferential cytidine-deaminase activity in motifs with a hierarchy CCC>CCC>CC (where the preferentially deaminated C is underlined), to develop an eA3G-BE with distinctive CC context-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3-58.0% and 54.5-92.2% with excellent CC context-specificity were generated in human cells and rabbit embryos, respectively. In addition, a base editor that can further recognize relaxed NG PAMs is achieved by combining hA3G with an engineered SpCas9-NG variant. The A3G-BEs were used to induce accurate single-base substitutions which led to nonsense mutation with an efficiency of 83-100% and few bystander mutations in Founder (F0) rabbits at Tyr loci. CONCLUSIONS: These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.
APOBEC-3G Deaminase/genetics , CRISPR-Associated Protein 9/genetics , Cytidine Deaminase/genetics , APOBEC-3G Deaminase/metabolism , Animals , Gene Editing , Humans , Rabbits
Cytidine base editors, composed of a cytidine deaminase fused to Cas9 nickase, enable efficient C-to-T conversion in various organisms. However, current base editors suffer from severe trade-off between editing efficiency and precision. Here, based on rationally mutated cytidine deaminase domain, we develop a new base editor, YFE-BE4max, effectively narrow the editing width to as little as approximately three nucleotides while maintaining high efficiency in rabbits. Moreover, YFE-BE4max successfully mediated the Tyr p. Q68Stop and Lmna p. G607G mutation in F0 rabbit with high efficiency and precision, which precisely recapitulates the pathological features of human OCA1 and HGPS, respectively. Collectively, YFE-BE4max system provide promising tools to perform efficient base editing with high precision in rabbits and enhances its capacity to precisely model human diseases.
Gene Editing , Albinism/genetics , Animals , Base Sequence , Cytidine Deaminase/chemistry , Cytidine Deaminase/genetics , Cytosine/metabolism , Disease Models, Animal , Embryo, Mammalian/metabolism , Lamin Type A/genetics , Mutation/genetics , Progeria/genetics , Protein Domains , Rabbits , Thymidine/genetics
Escherichia coli (E. coli) is one of the common pathogenic bacteria in veterinary clinical infection. As an opportunistic microorganism, E. coli normally does not cause diseases. However, it causes infections under certain circumstance to domesticated animal and poultry, resulting in severe diarrhea, septicemia, and respiratory infections. Although there are increasing reports regarding the infections of E. coli to domestic animals and poultry, the infection of E. coli in dogs is relatively less reported, especially on septicemia and meningoencephalitis. Here, we reported the isolation and identification of an E. coli isolate named CEC-GZL17 from dogs characterized by septicemia and sudden death, and found that CEC-GZL17 is able to cause meningoencephalitis. Exploration on the potential mechanism underlying meningoencephalitis demonstrated that CEC-GZL17 infection significantly increases TNF-α expression and inhibits ZO-1 and occludin expressions in brain tissue, indicating that the E coli likely use the mechanism to penetrate the blood-brain barrier via disrupting tight junction architecture, thus leading to the invasion to brain tissue.
Blood-Brain Barrier/microbiology , Escherichia coli Infections/veterinary , Escherichia coli/pathogenicity , Meningoencephalitis/pathology , Sepsis/pathology , Tight Junctions/microbiology , Animals , Blood-Brain Barrier/pathology , Brain/metabolism , Dog Diseases/microbiology , Dogs , Escherichia coli Infections/pathology , Meningoencephalitis/microbiology , Meningoencephalitis/veterinary , Mice , Occludin/biosynthesis , Sepsis/microbiology , Sepsis/veterinary , Tight Junctions/pathology , Tumor Necrosis Factor-alpha/biosynthesis , Zonula Occludens-1 Protein/biosynthesis
Poly(ethylene terephthalate) (PET) generally suffers from low crystallization rate and long molding duration, which as a result limit its application as engineering plastics. To overcome these drawbacks, series of PET/layered double hydroxide (LDH) nanocomposites were prepared by a solution blending process. The effect of metal composition (MgAl and CaAl) and organo-modification (stearic acid intercalated) for LDH fillers on the crystallization behavior of the nanocomposites was investigated. It was revealed that, compared with PET/CaAl-LDH, the PET/MgAl-LDH nanocomposite exhibits a higher crystallization temperature and faster crystallization rate, which is associated with the superior nucleation ability of MgAl-LDH. The nucleation mechanism of PET induced by LDHs was explored by means of Avrami equation and theory of Hoffman-Lauritzen, pointing out that the incorporation of LDHs reduce the free energy of nucleation and the fold surface free energy of PET. In order to improve the compatibility between LDH and PET, stearic acid (SA) intercalated MgAl-LDH was prepared and filled into PET matrix. The resultant PET/MgAl-LDH-SA shows a further enhanced crystallization temperature and accelerated crystallization rate, in comparison with PET/MgAl-LDH nanocomposites. In addition, the thermal stability, gas barrier and mechanical properties of PET/LDH composites were improved upon incorporation of LDH fillers.
Layered double hydroxide (LDH) platelets with nanosized and microsized level were synthesized and used as fillers in an isotactic polypropylene (PP) matrix. The nucleation and crystallization behavior of PP/LDH composites (denoted as 1-PPLx and 2-PPLx for composites containing nanosized and microsized LDH, respectively; x represents the mass percentage of LDH) was investigated by differential scanning calorimetry and polarized optical microscopy techniques. It is found that the crystallization temperature of PP/LDH composites is largely enhanced and the half crystallization time is reduced remarkably relative to pure PP, especially for 2-PPLx composite. The 2-PPLx composite exhibits stronger heterogeneous nucleating ability and faster crystallization rate than 1-PPLx samples with the same LDH loading. In addition, the crystallized PP/LDH composites possess significantly enhanced thermal stability, gas barrier, and flame-retardant properties relative to neat PP, which would show a broad application prospect in engineering plastics and packing industry.
