Precise large-fragment deletions in mammalian cells and mice generated by dCas9-controlled CRISPR/Cas3.

Currently, the Cas9 and Cas12a systems are widely used for genome editing, but their ability to precisely generate large chromosome fragment deletions is limited. Type I-E CRISPR mediates broad and unidirectional DNA degradation, but controlling the size of Cas3-mediated DNA deletions has proven elusive thus far. Here, we demonstrate that the endonuclease deactivation of Cas9 (dCas9) can precisely control Cas3-mediated large-fragment deletions in mammalian cells. In addition, we report the elimination of the Y chromosome and precise retention of the Sry gene in mice using CRISPR/Cas3 and dCas9-controlled CRISPR/Cas3, respectively. In conclusion, dCas9-controlled CRISPR/Cas3-mediated precise large-fragment deletion provides an approach for establishing animal models by chromosome elimination. This method also holds promise as a potential therapeutic strategy for treating fragment mutations or human aneuploidy diseases that involve additional chromosomes.

Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope.

BACKGROUND: Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. RESULTS: Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797-C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. CONCLUSIONS: These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.

Development of a highly efficient prime editor system in mice and rabbits.

The recently developed prime-editing (PE) technique is more precise than previously available techniques and permits base-to-base conversion, replacement, and insertions and deletions in the genome. However, previous reports show that the efficiency of prime editing is insufficient to produce genome-edited animals. In fact, prime-guide RNA (pegRNA) designs have posed a challenge in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 °C, leading to optimal performance in cells, and we found that the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated a Hoxd13 (c. 671 G > T) mutation in mice and a Tyr (c. 572 del) mutation in rabbits by ePE3max. Overall, the editing efficiency of modified ePE3max systems is superior to that of the original PE3 system in producing genome-edited animals, which can serve as an effective and versatile genome-editing tool for precise genome modification in animal models.

A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.

BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.

Highly efficient A-to-G base editing by ABE8.17 in rabbits.

Adenine base editors (ABEs), composed of an evolved adenine deaminase fused to the Cas9 nickase, enable efficient and precise A-to-G conversion in various organisms. However, the base editing of some challenging loci with the ABE7.10 system in rabbits was inefficient in our previous study. Here, we show that ABE8.17 and SpRY-ABE8.17 can efficiently induce base editing in mouse and rabbit embryos. In addition, this strategy can be used to precisely mimic clinical point mutations in rabbits. Furthermore, by eliminating the linker in ABE8.17, we created ABE8.17-NL, which achieved efficient base editing within a narrowed window (2-4 nts) in human HEK293FT cells. Collectively, these findings show that ABE8.17 systems can efficiently induce efficient A-to-G base editing at desired sites and that the ABE7.10 system is inefficient, thus providing an efficient way to generate ideal disease models in rabbits.

Live imaging of RNA and RNA splicing in mammalian cells via the dcas13a-SunTag-BiFC system.

Dynamic tracking of the localization of RNA molecules (nucleus and/or cytoplasm) and RNA splicing in living cells plays an important role in understanding their functions. However, a lack of dynamic imaging and high background fluorescence have been reported in the fluorescence in situ hybridization (FISH). Here, we developed a new tool, the dcas13a-SunTag-BiFC system, which fused the dLwacas13a and SunTag systems. dLwacas13a is used as a tracker to target specific RNAs, while SunTag recruits split Venus fluorescent proteins to label targeted RNAs. Our results showed that 4 × NLS-dCas13a-24 × SunTag-BiFC and 2 × NLS- dCas13a-24 × SunTag-BiFC systems can be used for imaging of endogenous RNA foci in the nucleus (Xist) and cytoplasm (Ppib and stress granules) in living cells, respectively. Compared to 12x MS2-MCP system, the dcas13a-SunTag-BiFC system showed a better performance of mRNA foci tracking in live cells. Furthermore, we confirmed the premature termination codon (PTC)-induced exon skipping of Oxt RNA using the dcas13a-SunTag-BiFC and MS2-MCP systems in the nucleus. Thus, the dcas13a-SunTag-BiFC system will facilitate the study of RNA localization in living cells and provide new insights into RNA translocation and splicing.

Association of genetic polymorphisms in immune-related lncRNA with osteoarthritis susceptibility in Chinese Han population.

AIM: Immune-related lncRNA may influence osteoarthritis (OA) susceptibility. We conducted this study to assess whether the genetic variants in several immune-related lncRNA influence OA susceptibility. METHODS: The current research genotyped four SNPs in 306 OA patients and 316 healthy controls, including PRNCR1 rs7463708, PRNCR1 rs1456315, PRNCR1 rs16901946 and KIF13B1 rs643472, to investigate their associations with OA susceptibility. RESULTS: We identified that PRNCR1 rs1456315 was associated with OA susceptibility in Chinese Han population (recessive model: odds ratio = 2.24; 95% CI: 1.05-4.81; additive model: odds ratio = 1.36; 95% CI: 1.03-1.80). CONCLUSION: Individuals with PRNCR1 rs1456315 mutant G allele were more likely to suffer from OA in Chinese Han population.

Follicle-stimulating hormone (FSH) promotes retinol uptake and metabolism in the mouse ovary.

BACKGROUND: Retinoids (retinol and its derivatives) are required for the development and maintenance of normal physiological functions of the ovary. However, the mechanisms underlying the regulation of ovarian retinoid homeostasis during follicular development remain unclear. METHODS: The present study determined retinoid levels and the expression levels of genes involved in the retinol uptake and its metabolic pathway in the ovaries of follicle-stimulating hormone (FSH)-treated mice and in granulosa cells treated with FSH using ultra performance liquid chromatography (UPLC) combined with quadrupole time-of-flight high-sensitivity mass spectrometry (Q-TOF/HSMS) and real-time PCR analysis. RESULTS: The levels of total retinoids and retinoic acid (RA) and expressions of retinol-oxidizing enzyme genes alcohol dehydrogenase 1 (Adh1) and aldehyde dehydrogenase (Aldh1a1) are increased in the ovaries of mice treated with FSH; in contrast, the retinyl ester levels and retinol-esterifying enzyme gene lecithin: retinol acyltransferase (Lrat) expression are diminished. In FSH-treated granulosa cells, the levels of retinyl esters, retinaldehyde, and total retinoids are augmented; and this is coupled with an increase in the expressions of stimulated by retinoic acid 6 (Stra6) and cellular retinol-binding protein 1 (Crbp1), genes in the retinol uptake pathway, and Adh1, Adh7, and Aldh1a1 as well as a diminution in Lrat expression. CONCLUSIONS: These data suggest that FSH promotes retinol uptake and its conversion to RA through modulating the pathways of retinol uptake and metabolism in the mouse ovary. The present study provides a possible mechanism for the regulation of endogenous RA signaling in the developing follicles.

Polyol-assisted vermiculite dispersion in polyurethane nanocomposites.

The largest use of polyurethane (PU) is as closed cell rigid foams for thermal insulation. One problem is loss of blowing gases, which leads to slow increase in thermal conductivity. PU composites with plate-like nanofillers create a diffusion barrier, reducing gas transport and slowing insulation aging. In this research, a new in situ intercalative polymerization is described to disperse vermiculite (VMT) in PU. When VMT was modified by cation exchange with long-chain quaternary ammonium, the dispersion in methylene diphenyl diisocyanate (MDI) was significantly improved. Dispersion of clay in MDI was further improved by combining high intensity dispersive mixing with a polyol-clay preblend (master-batch). The VMT dispersibility was characterized using rheology, microscopy, and X-ray scattering/diffraction. With the method of polyol-assisted VMT dispersion, electron microscopy revealed extensive intercalation and exfoliation of clay particles. In contrast, simple mixing of organoclay in MDI resulted in macroscopic localization and poor distribution of clay particles in PU. The final nanocomposites prepared by the master-batch method showed enhancement of mechanical properties (85% increase in elastic modulus) and reduction in permeability to CO2, as much as 40%, at a low clay concentration of 3.3 wt %.

Synthesis and properties of vermiculite-reinforced polyurethane nanocomposites.

Natural vermiculite was modified by cation exchange with long-chain quaternary alkylammonium salts and then dispersed in polyether-based polyols with different structures and ethylene oxide/propylene oxide ratios. The dispersions were evaluated by X-ray scattering and rheology. In all polyol dispersions tested, polyols were intercalated into the vermiculite interlayers. Also, significant shear thinning behavior was observed. A large interlayer spacing of ∼90 Šwas achieved in one polyol suitable for polyurethane elastomer synthesis. In polyurethane made with this polyol, clay platelets were extensively intercalated or exfoliated. The composites showed a >270% increase in tensile modulus, >60% increase in tensile strength, and a 30% reduction in N(2) permeability with a loading of 5.3 wt % clay in polyurethane. Differential scanning calorimetry and dynamic mechanical analysis revealed that the nanoclay interacts with the polyurethane hard segments.

Guidance of olfactory ensheathing cell growth and migration on electrospun silk fibroin scaffolds.

Transplantation of olfactory ensheathing cells (OECs) is a potential treatment for spinal cord injury (SCI). However, this process lacks extracellular matrix guiding cell growth, tissue morphogenesis, and remodeling. In order to solve this problem, we fabricated silk fibroin scaffolds (SFS) with different fiber diameters by electrospinning. The behaviors of OECs on 300 and 1800 nm SFS were studied by analyzing cell morphological feature, distribution, and proliferation. The results showed the 300 nm SFS with good potential to guide OECs growth. Subsequently, the properties of 300 nm SFS were further investigated along with PLL. With 300 nm SFS, the preservation of cell phenotype was confirmed by the presence of cell-specific markers, including nerve growth factor receptor p75 and glial fibrillary acidic protein. And the migration behaviors of OECs were also observed by Leica AF6000. In addition, migration tracks, turning behavior, migration distances, migration speeds, and forward migration indices were calculated. Furthermore, the expression of neurotrophic factors was assayed at transcription and protein levels using RT-PCR and ELISA. All these results indicated the diameter of the fiber played an important role in guiding cell adhesion, growth, and migration in vitro and the 300 nm SFS could be suitable to construct tissue-engineered scaffolds for SCI repair.

[Biocompatibility of silk fibroin nanofibers scaffold with olfactory ensheathing cells].

OBJECTIVE: To investigate the biocompatibility of silk fibroin nanofibers scaffold with olfactory ensheathing cells (OECs) and to provide an ideal tissue engineered scaffold for the repair of spinal cord injury (SCI). METHODS: Silk fibroin nanofibers were prepared using electrospinning techniques and were observed by scanning electron microscope (SEM). Freshly isolated OECs from SD rats purified by the modified differential adherent velocity method were cultured. The cells at passage 1 (1 x 10(4) cells/cm2) were seeded on the poly-l-lysine (control group) and the silk fibroin nanofibers (experimental group) coated coverslips in Petri dish. At desired time points, the morphological features, growth, and adhesion of the cells were observed using phase contrast inverted microscopy. The OECs were identified by the nerve growth factor receptor p75 (NGFR p75) immunofluorescence staining. The viability of OECs was examined by live/dead assay. The proliferation of OECs was examined by MTT assay. The cytotoxicity of the nanofibers was evaluated. RESULTS: The SEM micrographs showed that the nanofibers had a smooth surface with solid voids among the fibers, interconnecting a porous network, constituted a fibriform three dimensional structure and the average diameter of the fibers was about (260 +/- 84) nm. The morphology of OECs on the experimental group was similar to the cell morphology on the control group, the cells distributed along the fibers, and the directions of the cell protrusions were in the same as that of the fibers. Fluorescence microscopy showed that the purity of OECs was 74.21% +/- 2.48% in the experimental group and 79.05% +/- 2.52% in the control group 5 days after culture. There was no significant difference on cell purity between two groups (P > 0.05). The OECs in the experimental group stained positive for NGFR p75 compared to the control group, indicating that the cells in the experimental group still maintained the OECs characteristic phenotype. Live/dead staining showed that high viability was observed in both groups 3 days after culture. There was no significant difference on cell viability between two groups. The proliferation activity at 1, 3, 5, 7, and 10 days was examined by MTT assay. The absorbency values of the control group and the experimental group had significant differences 3 and 5 days after culture (P < 0.05). The relative growth rates were 95.11%, 90.35%, 92.63%, 94.12%, and 94.81%. The cytotoxicity of the material was grade 1 and nonvenomous according to GB/T 16886 standard. CONCLUSION: Silk fibroin nanofibers scaffold has good compatibility with OECs and is a promising tissue engineered scaffold for the repair of SCI.

Mesoporous concentric magnetic FePt core-shells nanoparticle with functionalized surfaces for capturing metal ions and DNA molecules.

It is demonstrated that monodisperse magnetic FePt nanoparticle can be engineered into a protective dense silica layer, followed by concentric outer mesoporous silica layers with tailored -SH, -SO3H and -NH2 surface groups, these new materials can be used to capture heavy metal ions and DNA molecules from solution specifically by their internal or/and external functionalised surfaces by magnetic means.