Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93 Gb (contig N50: 8.3 Mb, scaffold N50: 22.0 Mb, including 39.3 Mb N-bases), together with 206 Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8 Mb of HX1-specific sequences, including 4.1 Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations.

Aberrant activation of NF-κB is associated with the development of cancer and autoimmune and inflammatory diseases. IKKs are well recognized as key regulators in the NF-κB pathway and therefore represent attractive targets for intervention with small molecule inhibitors. Herein, we report that a complex natural product ainsliadimer A is a potent inhibitor of the NF-κB pathway. Ainsliadimer A selectively binds to the conserved cysteine 46 residue of IKKα/β and suppresses their activities through an allosteric effect, leading to the inhibition of both canonical and non-canonical NF-κB pathways. Remarkably, ainsliadimer A induces cell death of various cancer cells and represses in vivo tumour growth and endotoxin-mediated inflammatory responses. Ainsliadimer A is thus a natural product targeting the cysteine 46 of IKKα/β to block NF-κB signalling. Therefore, it has great potential for use in the development of anticancer and anti-inflammatory therapies.

Recent advances in the preparation of shape-shifting and size-growing nanostructures are hot topics in development of nanoscience, because many intelligent functions are always relied on their shape and dimension. Here we report a tunable manipulation of sequential self-assembled transformation in situ via a hierarchical assembly strategy based on a living thiol-disulfide exchange reaction. By tailoring the external stimuli, the reactive points can be generated at the ends of initially unimolecular micelles, which subsequently drive the pre-assemblies to periodically proceed into the hierarchically micellar connection, axial growth, bending, and cyclization processes from nanoscopic assemblies to macroscopic particles. Of particular interest would be systems that acquired the shape control and size adjustment of self-assemblies after termination or reactivation of disulfide reshuffling reaction by regulating external stimuli whenever needed. Such a hierarchical strategy for self-assembled evolution is universally applicable not only for other disulfide-linked dendritic polymers but also for exploitation of biological applications.

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.

Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly reduced from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.

DNA base editors, typically comprising editing enzymes fused to the N-terminus of nCas9, display off-target effects on DNA and/or RNA, which have remained an obstacle to their clinical applications. Off-target edits are typically countered via rationally designed point mutations, but the approach is tedious and not always effective. Here, we report that the off-target effects of both A > G and C > T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen. Furthermore, employing this Cas-embedding strategy, we have created a highly specific editor capable of efficient C > T editing at methylated and GC-rich sequences.

The type V-F CRISPR-Cas12f system is a strong candidate for therapeutic applications due to the compact size of the Cas12f proteins. In this work, we identify six uncharacterized Cas12f1 proteins with nuclease activity in mammalian cells from assembled bacterial genomes. Among them, OsCas12f1 (433 aa) from Oscillibacter sp. and RhCas12f1 (415 aa) from Ruminiclostridium herbifermentans, which respectively target 5' T-rich Protospacer Adjacent Motifs (PAMs) and 5' C-rich PAMs, show the highest editing activity. Through protein and sgRNA engineering, we generate enhanced OsCas12f1 (enOsCas12f1) and enRhCas12f1 variants, with 5'-TTN and 5'-CCD (D = not C) PAMs respectively, exhibiting much higher editing efficiency and broader PAMs, compared with the engineered variant Un1Cas12f1 (Un1Cas12f1_ge4.1). Furthermore, by fusing the destabilized domain with enOsCas12f1, we generate inducible-enOsCas12f1 and demonstate its activity in vivo by single adeno-associated virus delivery. Finally, dead enOsCas12f1-based epigenetic editing and gene activation can also be achieved in mammalian cells. This study thus provides compact gene editing tools for basic research with remarkable promise for therapeutic applications.

Influenza A virus (IAV) polymerase protein PB2 has been shown to partially inhibit the host immune response by blocking the induction of interferons (IFNs). However, the IAV PB2 protein that regulates the downstream signaling pathway of IFNs is not well characterized. Here, we report that IAV PB2 protein reduces cellular sensitivity to IFNs, suppressing the activation of STAT1/STAT2 and ISGs. Furthermore, IAV PB2 protein targets mammalian JAK1 at lysine 859 and 860 for ubiquitination and degradation. Notably, the H5 subtype of highly pathogenic avian influenza virus with I283M/K526R mutations on PB2 increases the ability to degrade mammalian JAK1 and exhibits higher replicate efficiency in mammalian (but not avian) cells and mouse lung tissues, and causes greater mortality in infected mice. Altogether, these data describe a negative regulatory mechanism involving PB2-JAK1 and provide insights into an evasion strategy from host antiviral immunity employed by IAV.

Inhibited immune response and low levels of delivery restrict starvation cancer therapy efficacy. Here, we report on the co-delivery of glucose oxidase (GOx) and indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyltryptophan using a metal-organic framework (MOF)-based nanoreactor, showing an amplified release for tumor starvation/oxidation immunotherapy. The nanosystem significantly overcomes the biobarriers associated with tumor penetration and improves the cargo bioavailability owing to the weakly acidic tumor microenvironment-activated charge reversal and size reduction strategy. The nanosystem rapidly disassembles and releases cargoes in response to the intracellular reactive oxygen species (ROS). GOx competitively consumes glucose and generates ROS, further inducing the self-amplifiable MOF disassembly and drug release. The starvation/oxidation combined IDO-blockade immunotherapy not only strengthens the immune response and stimulates the immune memory through the GOx-activated tumor starvation and recruitment of effector T cells, but also effectively relieves the immune tolerance by IDO blocking, remarkably inhibiting the tumor growth and metastasis in vivo.

Gut microbiota plays an important role in pig health and production. Still, availability of sequenced genomes and functional information for most pig gut microbes remains limited. Here we perform a landscape survey of the swine gut microbiome, spanning extensive sample sources by deep metagenomic sequencing resulting in an expanded gene catalog named pig integrated gene catalog (PIGC), containing 17,237,052 complete genes clustered at 90% protein identity from 787 gut metagenomes, of which 28% are unknown proteins. Using binning analysis, 6339 metagenome-assembled genomes (MAGs) were obtained, which were clustered to 2673 species-level genome bins (SGBs), among which 86% (2309) SGBs are unknown based on current databases. Using the present gene catalog and MAGs, we identified several strain-level differences between the gut microbiome of wild boars and commercial Duroc pigs. PIGC and MAGs provide expanded resources for swine gut microbiome-related research.

Irrigation is the largest sector of human water use and an important option for increasing crop production and reducing drought impacts. However, the potential for irrigation to contribute to global crop yields remains uncertain. Here, we quantify this contribution for wheat and maize at global scale by developing a Bayesian framework integrating empirical estimates and gridded global crop models on new maps of the relative difference between attainable rainfed and irrigated yield (ΔY). At global scale, ΔY is 34 ± 9% for wheat and 22 ± 13% for maize, with large spatial differences driven more by patterns of precipitation than that of evaporative demand. Comparing irrigation demands with renewable water supply, we find 30-47% of contemporary rainfed agriculture of wheat and maize cannot achieve yield gap closure utilizing current river discharge, unless more water diversion projects are set in place, putting into question the potential of irrigation to mitigate climate change impacts.

While nanoscale quantum emitters are effective tags for measuring biomolecular interactions, their utilities for applications that demand single-unit observations are limited by the requirements for large numerical aperture (NA) objectives, fluorescence intermittency, and poor photon collection efficiency resulted from omnidirectional emission. Here, we report a nearly 3000-fold signal enhancement achieved through multiplicative effects of enhanced excitation, highly directional extraction, quantum efficiency improvement, and blinking suppression through a photonic crystal (PC) surface. The approach achieves single quantum dot (QD) sensitivity with high signal-to-noise ratio, even when using a low-NA lens and an inexpensive optical setup. The blinking suppression capability of the PC improves the QDs on-time from 15% to 85% ameliorating signal intermittency. We developed an assay for cancer-associated miRNA biomarkers with single-molecule resolution, single-base mutation selectivity, and 10-attomolar detection limit. Additionally, we observed differential surface motion trajectories of QDs when their surface attachment stringency is altered by changing a single base in a cancer-specific miRNA sequence.

ASXL2 is frequently mutated in acute myeloid leukaemia patients with t(8;21). However, the roles of ASXL2 in normal haematopoiesis and the pathogenesis of myeloid malignancies remain unknown. Here we show that deletion of Asxl2 in mice leads to the development of myelodysplastic syndrome (MDS)-like disease. Asxl2-/- mice have an increased bone marrow (BM) long-term haematopoietic stem cells (HSCs) and granulocyte-macrophage progenitors compared with wild-type controls. Recipients transplanted with Asxl2-/- and Asxl2+/- BM cells have shortened lifespan due to the development of MDS-like disease or myeloid leukaemia. Paired daughter cell assays demonstrate that Asxl2 loss enhances the self-renewal of HSCs. Deletion of Asxl2 alters the expression of genes critical for HSC self-renewal, differentiation and apoptosis in Lin-cKit+ cells. The altered gene expression is associated with dysregulated H3K27ac and H3K4me1/2. Our study demonstrates that ASXL2 functions as a tumour suppressor to maintain normal HSC function.

Drug resistance and tumor recurrence are major challenges in cancer treatment. Cancer cells often display centrosome amplification. To maintain survival, cancer cells achieve bipolar division by clustering supernumerary centrosomes. Targeting centrosome clustering is therefore considered a promising therapeutic strategy. However, the regulatory mechanisms of centrosome clustering remain unclear. Here we report that KIFC1, a centrosome clustering regulator, is positively associated with tumor recurrence. Under DNA damaging treatments, the ATM and ATR kinases phosphorylate KIFC1 at Ser26 to selectively maintain the survival of cancer cells with amplified centrosomes via centrosome clustering, leading to drug resistance and tumor recurrence. Inhibition of KIFC1 phosphorylation represses centrosome clustering and tumor recurrence. This study identified KIFC1 as a prognostic tumor recurrence marker, and revealed that tumors can acquire therapeutic resistance and recurrence via triggering centrosome clustering under DNA damage stresses, suggesting that blocking KIFC1 phosphorylation may open a new vista for cancer therapy.

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19), with macrophages as one of the main cell types involved. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. Here, we establish a lung and macrophage co-culture system derived from human pluripotent stem cells (hPSCs), modeling the host-pathogen interaction in SARS-CoV-2 infection. We find that both classically polarized macrophages (M1) and alternatively polarized macrophages (M2) have inhibitory effects on SARS-CoV-2 infection. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Moreover, M1 macrophages suppress the growth and enhance apoptosis of lung cells. Inhibition of viral entry using an ACE2 blocking antibody substantially enhances the activity of M2 macrophages. Our studies indicate differential immune response patterns in distinct macrophage phenotypes, which could lead to a range of COVID-19 disease severity.

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.

Shading in combination with extended photoperiods can cause exaggerated stem elongation (ESE) in soybean, leading to lodging and reduced yields when planted at high-density in high-latitude regions. However, the genetic basis of plant height in adaptation to these regions remains unclear. Here, through a genome-wide association study, we identify a plant height regulating gene on chromosome 13 (PH13) encoding a WD40 protein with three main haplotypes in natural populations. We find that an insertion of a Ty1/Copia-like retrotransposon in the haplotype 3 leads to a truncated PH13H3 with reduced interaction with GmCOP1s, resulting in accumulation of STF1/2, and reduced plant height. In addition, PH13H3 allele has been strongly selected for genetic improvement at high latitudes. Deletion of both PH13 and its paralogue PHP can prevent shade-induced ESE and allow high-density planting. This study provides insights into the mechanism of shade-resistance and offers potential solutions for breeding high-yielding soybean cultivar for high-latitude regions.

Interferometric scattering microscopy is increasingly employed in biomedical research owing to its extraordinary capability of detecting nano-objects individually through their intrinsic elastic scattering. To significantly improve the signal-to-noise ratio without increasing illumination intensity, we developed photonic resonator interferometric scattering microscopy (PRISM) in which a dielectric photonic crystal (PC) resonator is utilized as the sample substrate. The scattered light is amplified by the PC through resonant near-field enhancement, which then interferes with the <1% transmitted light to create a large intensity contrast. Importantly, the scattered photons assume the wavevectors delineated by PC's photonic band structure, resulting in the ability to utilize a non-immersion objective without significant loss at illumination density as low as 25 W cm-2. An analytical model of the scattering process is discussed, followed by demonstration of virus and protein detection. The results showcase the promise of nanophotonic surfaces in the development of resonance-enhanced interferometric microscopies.

Both low soil water content (SWC) and high atmospheric dryness (vapor pressure deficit, VPD) can negatively affect terrestrial gross primary production (GPP). The sensitivity of GPP to soil versus atmospheric dryness is difficult to disentangle, however, because of their covariation. Using global eddy-covariance observations, here we show that a decrease in SWC is not universally associated with GPP reduction. GPP increases in response to decreasing SWC when SWC is high and decreases only when SWC is below a threshold. By contrast, the sensitivity of GPP to an increase of VPD is always negative across the full SWC range. We further find canopy conductance decreases with increasing VPD (irrespective of SWC), and with decreasing SWC on drier soils. Maximum photosynthetic assimilation rate has negative sensitivity to VPD, and a positive sensitivity to decreasing SWC when SWC is high. Earth System Models underestimate the negative effect of VPD and the positive effect of SWC on GPP such that they should underestimate the GPP reduction due to increasing VPD in future climates.

RIG-I-MAVS signaling pathway plays a crucial role in defending against pathogen infection and maintaining immune balance. Upon detecting viral RNA, RIG-I triggers the formation of prion-like aggregates of the adaptor protein MAVS, which then activates the innate antiviral immune response. However, the mechanisms that regulate the aggregation of MAVS are not yet fully understood. Here, we identified WDR77 as a MAVS-associated protein, which negatively regulates MAVS aggregation. WDR77 binds to MAVS proline-rich region through its WD2-WD3-WD4 domain and inhibits the formation of prion-like filament of recombinant MAVS in vitro. In response to virus infection, WDR77 is recruited to MAVS to prevent the formation of its prion-like aggregates and thus downregulate RIG-I-MAVS signaling in cells. WDR77 deficiency significantly potentiates the induction of antiviral genes upon negative-strand RNA virus infections, and myeloid-specific Wdr77-deficient mice are more resistant to RNA virus infection. Our findings reveal that WDR77 acts as a negative regulator of the RIG-I-MAVS signaling pathway by inhibiting the prion-like aggregation of MAVS to prevent harmful inflammation.