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Tomato rhizosphere microbiome alterations that contribute to bacterial wilt resistance are detected using metagenomics. Tomato variety Hawaii 7996 is resistant to the soil-borne pathogen Ralstonia solanacearum , whereas the Moneymaker variety is susceptible to the pathogen. To evaluate whether plant-associated microorganisms have a role in disease resistance, we analyzed the rhizosphere microbiomes of both varieties in a mesocosm experiment. Microbiome structures differed between the two cultivars. Transplantation of rhizosphere microbiota from resistant plants suppressed disease symptoms in susceptible plants. Comparative analyses of rhizosphere metagenomes from resistant and susceptible plants enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plant. We cultivated this flavobacterium, named TRM1, and found that it could suppress R. solanacearum -disease development in a susceptible plant in pot experiments. Our findings reveal a role for native microbiota in protecting plants from microbial pathogens, and our approach charts a path toward the development of probiotics to ameliorate plant diseases.

O-antigens of Gram-negative bacteria modulate the interactions of bacterial cells with diverse external factors, including the components of the immune system and bacteriophages. Some phages need to acquire specific adhesins to overcome the O-antigen layer. For other phages, O-antigen is required for phage infection. In this case, interaction of phage receptor binding proteins coupled with enzymatic degradation or modification of the O-antigen is followed by phage infection. Identification of the strategies used by newly isolated phages may be of importance in their consideration for various applications. Here we describe an approach based on screening for host LPS alterations caused by selection by bacteriophages. We describe an optimized LPS profiling procedure that is simple, rapid and suitable for mass screening of mutants. We demonstrate that the phage infection strategies identified using a set of engineered E. coli 4 s mutants with impaired or altered LPS synthesis are in good agreement with the results of simpler tests based on LPS profiling of phage-resistant spontaneous mutants.

Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies 1 – 7 . However, CAR-T cell therapy currently has several limitations 8 – 12 . Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR–Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR + cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1 -integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy. Non-viral CAR-T cells with gene-specific targeted integration are safe and effective in patients with lymphoma.

Nucleic acid detection powered by CRISPR technology provides a rapid, sensitive, and deployable approach to molecular diagnostics. While exciting, there remain challenges limiting its practical applications, such as the need for pre-amplification and the lack of quantitative ability. Here, we develop an asymmetric CRISPR assay for cascade signal amplification detection of nucleic acids by leveraging the asymmetric trans -cleavage behavior of competitive crRNA. We discover that the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a can induce cascade signal amplification, significantly improving the target detection signal. In addition, we find that CRISPR-Cas12a can recognize fragmented RNA/DNA targets, enabling direct RNA detection by Cas12a. Based on these findings, we apply our asymmetric CRISPR assay to quantitatively detect microRNA without the need for pre-amplification, achieving a detection sensitivity of 856 aM. Moreover, using this method, we analyze and quantify miR-19a biomarker in plasma samples from bladder cancer patients. This asymmetric CRISPR assay has the potential to be widely applied for simple and sensitive nucleic acid detection in various diagnostic settings. New strategies are being developed to simplify CRISPR-based nucleic acid detection. By investigating the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a, the authors develop an asymmetric CRISPR assay for amplification-free, cascade signal amplification detection of nucleic acids.

The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3′- or 5′-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40–60 min. CRISPR-Cas12a based detection systems can be sensitive down to the picomolar range. Here the authors modify the 3′- and 5′-ends of the crRNA and show this enhances trans-cleavage for improved sensitivity.

Species often include multiple ecotypes that are adapted to different environments 1 . However, it is unclear how ecotypes arise and how their distinctive combinations of adaptive alleles are maintained despite hybridization with non-adapted populations 2 – 4 . Here, by resequencing 1,506 wild sunflowers from 3 species ( Helianthus annuus , Helianthus petiolaris and Helianthus argophyllus ), we identify 37 large (1–100 Mbp in size), non-recombining haplotype blocks that are associated with numerous ecologically relevant traits, as well as soil and climate characteristics. Limited recombination in these haplotype blocks keeps adaptive alleles together, and these regions differentiate sunflower ecotypes. For example, haplotype blocks control a 77-day difference in flowering between ecotypes of the silverleaf sunflower H. argophyllus (probably through deletion of a homologue of FLOWERING LOCUS T ( FT )), and are associated with seed size, flowering time and soil fertility in dune-adapted sunflowers. These haplotypes are highly divergent, frequently associated with structural variants and often appear to represent introgressions from other—possibly now-extinct—congeners. These results highlight a pervasive role of structural variation in ecotypic adaptation. Resequencing analyses of three species of wild sunflower identify large non-recombining haplotype blocks that correlate with ecologically relevant traits, soil and climate characteristics, and that differentiate species ecotypes.

Artificially improving traits of cultivated alfalfa ( Medicago sativa L.), one of the most important forage crops, is challenging due to the lack of a reference genome and an efficient genome editing protocol, which mainly result from its autotetraploidy and self-incompatibility. Here, we generate an allele-aware chromosome-level genome assembly for the cultivated alfalfa consisting of 32 allelic chromosomes by integrating high-fidelity single-molecule sequencing and Hi-C data. We further establish an efficient CRISPR/Cas9-based genome editing protocol on the basis of this genome assembly and precisely introduce tetra-allelic mutations into null mutants that display obvious phenotype changes. The mutated alleles and phenotypes of null mutants can be stably inherited in generations in a transgene-free manner by cross pollination, which may help in bypassing the debate about transgenic plants. The presented genome and CRISPR/Cas9-based transgene-free genome editing protocol provide key foundations for accelerating research and molecular breeding of this important forage crop. Alfalfa is an important forage crop, but genetic improvement is challenging due to the lack of a reference genome and an efficient genome editing protocol. Here, the authors report the chromosome-level assembly of the autotetraploid genome and a CRISPR/Cas9-based transgene-free genome editing protocol.

Metagenomic and biochemical analyses of soil samples from Antarctic desert regions provides evidence that bacteria in these soils derive carbon and energy from atmospheric CO, H 2 and CO 2 . Microbes use trace gases for energy Terrestrial Antarctica is one of the most extreme environments on Earth, but previous work has shown that it does support microbial life. How microbial communities sustain their energy and carbon needs at sites at which photosynthetic potential is low has been unclear. Belinda Ferrari and colleagues applied shotgun metagenomics and biochemical analyses to clarify the basis of primary production in two such Antarctic sites, and demonstrate that the atmospheric trace gases H 2 and CO are oxidized at rates sufficient to sustain these communities. This represents a novel mode of primary production, but further work is required to evaluate whether it is a widespread pathway for energy generation. Cultivation-independent surveys have shown that the desert soils of Antarctica harbour surprisingly rich microbial communities 1 , 2 , 3 . Given that phototroph abundance varies across these Antarctic soils 2 , 4 , an enduring question is what supports life in those communities with low photosynthetic capacity 3 , 5 . Here we provide evidence that atmospheric trace gases are the primary energy sources of two Antarctic surface soil communities. We reconstructed 23 draft genomes from metagenomic reads, including genomes from the candidate bacterial phyla WPS-2 and AD3. The dominant community members encoded and expressed high-affinity hydrogenases, carbon monoxide dehydrogenases, and a RuBisCO lineage known to support chemosynthetic carbon fixation 6 , 7 . Soil microcosms aerobically scavenged atmospheric H 2 and CO at rates sufficient to sustain their theoretical maintenance energy and mediated substantial levels of chemosynthetic but not photosynthetic CO 2 fixation. We propose that atmospheric H 2 , CO 2 and CO provide dependable sources of energy and carbon to support these communities, which suggests that atmospheric energy sources can provide an alternative basis for ecosystem function to solar or geological energy sources 8 , 9 . Although more extensive sampling is required to verify whether this process is widespread in terrestrial Antarctica and other oligotrophic habitats, our results provide new understanding of the minimal nutritional requirements for life and open the possibility that atmospheric gases support life on other planets.

The B.1.1.7 variant (also known as Alpha) of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the UK in the summer of 2020. The prevalence of this variant increased rapidly owing to an increase in infection and/or transmission efficiency 1 . The Alpha variant contains 19 nonsynonymous mutations across its viral genome, including 8 substitutions or deletions in the spike protein that interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that of the 8 individual spike protein substitutions, only N501Y resulted in consistent fitness gains for replication in the upper airway in a hamster model as well as in primary human airway epithelial cells. The N501Y substitution recapitulated the enhanced viral transmission phenotype of the eight mutations in the Alpha spike protein, suggesting that it is a major determinant of the increased transmission of the Alpha variant. Mechanistically, the N501Y substitution increased the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil, South Africa and elsewhere 2 , 3 , our results indicate that N501Y substitution is an adaptive spike mutation of major concern. Experiments in a hamster model of COVID-19 and human airway epithelial cells show that the spike N501Y mutation is the major determinant of increased fitness of the B.1.1.7 Alpha variant of SARS-CoV-2.

Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell cycle, instating a barrier for adult heart regeneration 1 , 2 . Here, to explore whether metabolic reprogramming can overcome this barrier and enable heart regeneration, we abrogate fatty acid oxidation in cardiomyocytes by inactivation of Cpt1b . We find that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia–reperfusion injury. Metabolic studies reveal profound changes in energy metabolism and accumulation of α-ketoglutarate in Cpt1b -mutant cardiomyocytes, leading to activation of the α-ketoglutarate-dependent lysine demethylase KDM5 (ref.  3 ). Activated KDM5 demethylates broad H3K4me3 domains in genes that drive cardiomyocyte maturation, lowering their transcription levels and shifting cardiomyocytes into a less mature state, thereby promoting proliferation. We conclude that metabolic maturation shapes the epigenetic landscape of cardiomyocytes, creating a roadblock for further cell divisions. Reversal of this process allows repair of damaged hearts. Inhibition of the fatty acid oxidation metabolic pathway through inactivation of Cpt1b enhances cardiomyocyte survival and proliferation and allows heart regeneration in adult mice.