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Ultrathin, molecular sieving membranes composed of microporous materials offer great potential to realize high permeances and selectivities in separation applications, but strategies for their production have remained a challenge. Here we show a route for the scalable production of nanometre-thick metal–organic framework (MOF) molecular sieving membranes, specifically via gel–vapour deposition, which combines sol–gel coating with vapour deposition for solvent-/modification-free and precursor-/time-saving synthesis. The uniform MOF membranes thus prepared have controllable thicknesses, down to ~17 nm, and show one to three orders of magnitude higher gas permeances than those of conventional membranes, up to 215.4 × 10 −7  mol m −2  s −1  Pa −1 for H 2 , and H 2 /C 3 H 8 , CO 2 /C 3 H 8 and C 3 H 6 /C 3 H 8 selectivities of as high as 3,400, 1,030 and 70, respectively. We further demonstrate the in situ scale-up processing of a MOF membrane module (30 polymeric hollow fibres with membrane area of 340 cm 2 ) without deterioration in selectivity. MOF-based membranes have shown great promise in separation applications, but producing thin membranes that allow for high fluxes remains challenging. Here, the authors use a gel–vapour deposition strategy to fabricate composite membranes with less than 20 nm thicknesses and high gas permeances and selectivities.

Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53–88% and 44–100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit. Base editors can make targeted changes without inducing a double-stranded break. Here, the authors apply the BE3 and ABE7.10 systems to rabbit to create highly efficient targeted base substitutions and various mutation types, and show reduced frequency of undesired by-products with the updated BE4-Gam system.

MgO nanoparticles have good As-adsorption capacity in treating As-contaminated wastewater but suffer from high production cost. In this study, instead of using pre-formed MgO nanoparticles, we found that in-situ formed Mg(OH) 2 from MgCl 2 and NaOH reaction exhibited super high arsenate (As(V)) removal efficiency. Only 1.5 mmol/L of in-situ formed Mg(OH) 2 could remove more than 95% As(V) within 10 min to make the As contaminated water (10 mg-As(V)/L) meet the municipal wastewater treatment standard, whereas MgO nanoparticles failed. The Mg-As sludge has an amorphous crystal structure while no Mg(OH) 2 phase could be observed. As(V) existed uniformly within the sludge which was confirmed by elemental mapping. A precipitation-adsorption-coagulation mechanism might exist, which could relieve the restriction of limited surface area of solid MgO adsorbents. This study not only reveals an applicable method for efficient removal of trace level As(V) from water but also implies the huge potential of in-situ formed adsorbents in water treatment.

Persistent luminescence nanoparticles （PLNPs） and upconversion nanoparticles （UCNPs） are two special optical imaging nanoprobes. In this study, efficient upconverted persistent luminescence （UCPL） is realized by combining their unique features into polymethyl methacrylate, forming a film composed of both PLNPs and UCNPs. The red persistent luminescence （-640 nm） of the PLNPs （CaS：Eu,Tm, Ce） can be activated by upconverted green emission of UCNPs （-NaYF4：Yb, Er@NaYF4） excited by near-infrared light （NIR）. Using this strategy, both the unique optical properties of PLNPs and UCNPs can be optimally synergized, thus generating efficient upconversion, photoluminescence, and UCPL simultaneously. The UCPL system has potential applications in in vivo bioimaging by simply monitoring the biocompatible low power density of NIR-light-excited persistent luminescence. Due to its simplicity, we anticipate that this method for the preparation of UCPL composite can be easily adjusted using other available upconversion and persistent phosphor pairs for a number of biophotonic and photonic applications.

The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications. Small Cas enzymes are required for therapeutic use. Here the authors report an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturisation and use this to generate five compact variants of Cas13 based on a combination of IDC strategy and AlphaFold2.

CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models. Myostatin (MSTN) is a negative regulator of muscle mass, related to muscle growth and differentiation. The knockout of MSTN with the desired phenotype of double muscle has been successfully generated in mice, goats, pigs and cattle, but not in rabbits. In this study, the MSTN knockout (KO) rabbits were generated by co-injection of Cas9 mRNA and sgRNA into zygotes. The typical phenotype of double muscle with hyperplasia or hypertrophy of muscle fiber was observed in MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation, suggesting that the mutation of MSTN could be stably inherited in the MSTN KO rabbits. In summary, we have successfully generated MSTN KO rabbits using CRISPR/Cas9 system with high efficiency, which is a reliable and effective animal model for the study of muscle development and related diseases.

Cytosine base editors (CBEs), combining cytidine deaminases with the Cas9 nickase (nCas9), enable targeted C-to-T conversions in genomic DNA and are powerful genome-editing tools used in biotechnology and medicine. However, the overexpression of cytidine deaminases in vivo leads to unexpected potential safety risks, such as Cas9-independent off-target effects. This risk makes the development of deaminase off switches for modulating CBE activity an urgent need. Here, we report the repurpose of four virus-derived anti-deaminases (Ades) that efficiently inhibit APOBEC3 deaminase-CBEs. We demonstrate that they antagonize CBEs by inhibiting the APOBEC3 catalytic domain, relocating the deaminases to the extranuclear region or degrading the whole CBE complex. By rationally engineering the deaminase domain, other frequently used base editors, such as CGBE, A&CBE, A&CGBE, rA1-CBE and ABE8e, can be moderately inhibited by Ades, expanding the scope of their applications. As a proof of concept, the Ades in this study dramatically decrease both Cas9-dependent and Cas9-independent off-target effects of CBEs better than traditional anti-CRISPRs (Acrs). Finally, we report the creation of a cell type-specific CBE-ON switch based on a microRNA-responsive Ade vector, showing its practicality. In summary, these natural deaminase-specific Ades are tools that can be used to regulate the genome-engineering functions of BEs. Anti-deaminases can inhibit APOBEC3, a component of cytosine base editors. Here Zhanjun Li and colleagues repurposed anti-deaminase proteins derived from viruses to inhibit base editors for use in efficient regulation of base editors’ activity in gene modification and therapeutic applications.

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research.

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca2+ oscillations and Ca2+-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca2+-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca2+-modulated activities with tailored function. Optogenetics is a technique that has been used to study nerve cells for several years. It involves genetically engineering these cells to produce proteins from light-sensitive bacteria, and results in nerve cells that will either send, or stop sending, nerve impulses when they are exposed to a particular color of light. Neuroscientists have learned a lot about brain circuits using the technique, and now researchers in many other fields are giving it a try. There are, however, several challenges to using optogenetics in other types of cells. Nerve cells create a tiny electrical impulses when they are activated, which helps them quickly transmit messages. But other types of cells use more diverse means to communicate and transmit signals. This means that optogenetics techniques must be adapted. Additionally, many cells are located deep in the body and so getting the light to them can be difficult. He, Zhang et al. have now developed an optogenetic system (termed “Opto-CRAC”) that can control immune cells buried deep in tissue. The action of immune cells can be tuned by controlling the flow of calcium ions through gate-like proteins in their membranes. He, Zhang et al. genetically engineered immune cells so that a calcium gate-controlling protein became light sensitive. When the cells were exposed to a blue light the calcium ion gates opened. When the light was turned off, the gates closed. More intense light caused more calcium to enter into the cells. Further experiments then revealed that exposing these engineered immune cells to blue light in the laboratory could trigger an immune response. The next obstacle was getting light to immune cells in a live animal. So, He, Zhang et al. used specific nanoparticles that have been shown to help transmit light deep within tissue. In these experiments, mice were injected with the light-sensitive immune cells and the nanoparticles. Then, a near-infrared laser beam that can transmit into the tissues was pointed at the mice. This caused calcium channels to open in the engineered cells deep in the mice. Finally, further experiments were used to show that this light-based stimulation could boost an immune response to aid the killing of cancer cells. Other scientists will likely use the technique to help them study immune, heart, and other types of cells that use calcium to communicate.

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 CC C >C C C>C C (where the preferentially deaminated C is underlined), to develop an eA3G-BE with distinctive C C context-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3–58.0% and 54.5–92.2% with excellent C C 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 C C context-specificity will expand the toolset for precise gene modification in organisms.