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Naturally evolved enzymes, despite their astonishingly large variety and functional diversity, operate predominantly through thermochemical activation. Integrating prominent photocatalysis modes into proteins, such as triplet energy transfer, could create artificial photoenzymes that expand the scope of natural biocatalysis 1 – 3 . Here, we exploit genetically reprogrammed, chemically evolved photoenzymes embedded with a synthetic triplet photosensitizer that are capable of excited-state enantio-induction 4 – 6 . Structural optimization through four rounds of directed evolution afforded proficient variants for the enantioselective intramolecular [2+2]-photocycloaddition of indole derivatives with good substrate generality and excellent enantioselectivities (up to 99% enantiomeric excess). A crystal structure of the photoenzyme–substrate complex elucidated the non-covalent interactions that mediate the reaction stereochemistry. This study expands the energy transfer reactivity 7 – 10 of artificial triplet photoenzymes in a supramolecular protein cavity and unlocks an integrated approach to valuable enantioselective photochemical synthesis that is not accessible with either the synthetic or the biological world alone. Triplet photoenzymes developed through genetic encoding and directed evolution result in excited-state photocatalysts that provide a valuable approach to enantioselective photochemical synthesis.

Misfolded proteins can be degraded by selective autophagy. The prevailing view is that ubiquitin-tagged misfolded proteins are assembled into aggregates by the scaffold protein p62, and the aggregates are then engulfed and degraded by autophagosomes. Here we report that p62 forms droplets in vivo which have liquid-like properties such as high sphericity, the ability to undergo fusion, and recovery after photobleaching. Recombinant p62 does not undergo phase separation in vitro; however, adding a K63 polyubiquitin chain to p62 induces p62 phase separation, which results in enrichment of high-molecular weight ubiquitin signals in p62 droplets. Mixing recombinant p62 with cytosol from p62 −/− cells also results in p62 phase separation in a polyubiquitination-dependent manner. Mechanistically, p62 phase separation is dependent on p62 polymerization, the interaction between p62 and ubiquitin, and the valence of the polyubiquitin chain. Moreover, p62 phase separation can be regulated by post-translational modifications such as phosphorylation. Finally, we demonstrate that disease-associated mutations in p62 can affect phase separation. We propose that polyubiquitin chain-induced p62 phase separation drives autophagic cargo concentration and segregation.

Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes ( Sp ) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum . Here we report a Francisella novicida ( Fn ) CRISPR-Cpf1-based genome-editing method for C. glutamicum . CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86–100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of Fn Cpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3 N +4 or 3 N +2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L -proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system. Corynebacterium glutamicum is an important industrial microbe, however it has proven difficult to genetically engineer using Cas9 from Streptococcus pyogenes . Here the authors report effective genome engineering of the bacterium using Cpf1 from Francisella novicida .

Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization 1 – 5 . First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties. The BRAIN Initiative Cell Census Network has constructed a multimodal cell census and atlas of the mammalian primary motor cortex in a landmark effort towards understanding brain cell-type diversity, neural circuit organization and brain function.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca 2+ concentration, which activate Ca 2+ -binding proteins and upregulate the Na + /H + antiporter in order to remove Na + . Salt-induced increases in Ca 2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca 2+ -imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca 2+] i increases 1 ( moca1 ), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca 2+ spikes and waves, Na + /H + antiporter activation, and regulation of growth. Na + binds to GIPCs to gate Ca 2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops. The sphingolipid GIPC in the plant cell plasma membrane binds to sodium and triggers calcium influx, thereby triggering responses to excess salt such as efflux of sodium ions from cells.

Disruption of susceptibility ( S ) genes in crops is an attractive breeding strategy for conferring disease resistance 1 , 2 . However, S genes are implicated in many essential biological functions and deletion of these genes typically results in undesired pleiotropic effects 1 . Loss-of-function mutations in one such S gene, Mildew resistance locus O ( MLO ), confers durable and broad-spectrum resistance to powdery mildew in various plant species 2 , 3 . However, mlo- associated resistance is also accompanied by growth penalties and yield losses 3 , 4 , thereby limiting its widespread use in agriculture. Here we describe Tamlo-R32 , a mutant with a 304-kilobase pair targeted deletion in the MLO-B1 locus of wheat that retains crop growth and yields while conferring robust powdery mildew resistance. We show that this deletion results in an altered local chromatin landscape, leading to the ectopic activation of Tonoplast monosaccharide transporter 3 ( TaTMT3B ), and that this activation alleviates growth and yield penalties associated with MLO disruption. Notably, the function of TMT3 is conserved in other plant species such as Arabidopsis thaliana . Moreover, precision genome editing facilitates the rapid introduction of this mlo resistance allele ( Tamlo-R32) into elite wheat varieties. This work demonstrates the ability to stack genetic changes to rescue growth defects caused by recessive alleles, which is critical for developing high-yielding crop varieties with robust and durable disease resistance. Tamlo-R32 , an engineered wheat mutant allele of the Mildew resistance locus O ( MLO ) gene, confers resistance to powdery mildew, retains robust wheat growth, and can be transferred to other agriculturally important wheat varieties.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A 2A receptor (A 2A R). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A 2A R with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A 2A R are superior to shRNA mediated knockdown or pharmacological blockade of A 2A R. Mechanistically, human A 2A R-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A 2A R deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A 2A R for the improvement of CAR T cell function in the clinic. Activation of the adenosine receptor A2AR is associated with suppression of T cell function in the tumor microenvironment. To overcome immunosuppression, here the authors show that CRISPR/Cas9 mediated deletion of A2AR enhances CAR T cell effector functions without altering memory or persistence properties, improving CAR-T mediated tumor control in pre-clinical models.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex. Single-cell transcriptomics of more than 20,000 cells from two functionally distinct areas of the mouse neocortex identifies 133 transcriptomic types, and provides a foundation for understanding the diversity of cortical cell types.

The intensive application of inorganic nitrogen underlies marked increases in crop production, but imposes detrimental effects on ecosystems 1 , 2 : it is therefore crucial for future sustainable agriculture to improve the nitrogen-use efficiency of crop plants. Here we report the genetic basis of nitrogen-use efficiency associated with adaptation to local soils in rice ( Oryza sativa L.). Using a panel of diverse rice germplasm collected from different ecogeographical regions, we performed a genome-wide association study on the tillering response to nitrogen—the trait that is most closely correlated with nitrogen-use efficiency in rice—and identified OsTCP19 as a modulator of this tillering response through its transcriptional response to nitrogen and its targeting to the tiller-promoting gene DWARF AND LOW-TILLERING ( DLT ) 3 , 4 . A 29-bp insertion and/or deletion in the OsTCP19 promoter confers a differential transcriptional response and variation in the tillering response to nitrogen among rice varieties. The allele of OsTCP19 associated with a high tillering response to nitrogen is prevalent in wild rice populations, but has largely been lost in modern cultivars: this loss correlates with increased local soil nitrogen content, which suggests that it might have contributed to geographical adaptation in rice. Introgression of the allele associated with a high tillering response into modern rice cultivars boosts grain yield and nitrogen-use efficiency under low or moderate levels of nitrogen, which demonstrates substantial potential for rice breeding and the amelioration of negative environment effects by reducing the application of nitrogen to crops. OsTCP19 is a modulator of the tillering response to nitrogen in rice, and introgression of an allele of OsTCP19 associated with a high tillering response into modern rice cultivars markedly improves their nitrogen-use efficiency.

Retrotransposable elements are deleterious at many levels, and the failure of host surveillance systems for these elements can thus have negative consequences. However, the contribution of retrotransposon activity to ageing and age-associated diseases is not known. Here we show that during cellular senescence, L1 (also known as LINE-1) retrotransposable elements become transcriptionally derepressed and activate a type-I interferon (IFN-I) response. The IFN-I response is a phenotype of late senescence and contributes to the maintenance of the senescence-associated secretory phenotype. The IFN-I response is triggered by cytoplasmic L1 cDNA, and is antagonized by inhibitors of the L1 reverse transcriptase. Treatment of aged mice with the nucleoside reverse transcriptase inhibitor lamivudine downregulated IFN-I activation and age-associated inflammation (inflammaging) in several tissues. We propose that the activation of retrotransposons is an important component of sterile inflammation that is a hallmark of ageing, and that L1 reverse transcriptase is a relevant target for the treatment of age-associated disorders. During cellular senescence in human and mouse cells, L1 transposons become transcriptionally derepressed and trigger a type-1 interferon response, which contributes to age-associated inflammation and age-related phenotypes.