Genome-wide DNase I-hypersensitive site assay reveals distinct genomic distributions and functional features of open chromatin in autopolyploid sugarcane.

The characterization of cis-regulatory DNA elements (CREs) is essential for deciphering the regulation of gene expression in eukaryotes. Although there have been endeavors to identify CREs in plants, the properties of CREs in polyploid genomes are still largely unknown. Here, we conducted the genome-wide identification of DNase I-hypersensitive sites (DHSs) in leaf and stem tissues of the auto-octoploid species Saccharum officinarum. We revealed that DHSs showed highly similar distributions in the genomes of these two S. officinarum tissues. Notably, we observed that approximately 74% of DHSs were located in distal intergenic regions, suggesting considerable differences in the abundance of distal CREs between S. officinarum and other plants. Leaf- and stem-dependent transcriptional regulatory networks were also developed by mining the binding motifs of transcription factors (TFs) from tissue-specific DHSs. Four TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF1 (TCP) TFs (TCP2, TCP4, TCP7, and TCP14) and two ethylene-responsive factors (ERFs) (ERF109 and ERF03) showed strong causal connections with short binding distances from each other, pointing to their possible roles in the regulatory networks of leaf and stem development. Through functional validation in transiently transgenic protoplasts, we isolate a set of tissue-specific promoters. Overall, the DHS maps presented here offer a global view of the potential transcriptional regulatory elements in polyploid sugarcane and can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding.

Genome-wide chromatin accessibility analysis unveils open chromatin convergent evolution during polyploidization in cotton.

Allopolyploidization, resulting in divergent genomes in the same cell, is believed to trigger a "genome shock", leading to broad genetic and epigenetic changes. However, little is understood about chromatin and gene-expression dynamics as underlying driving forces during allopolyploidization. Here, we examined the genome-wide DNase I-hypersensitive site (DHS) and its variations in domesticated allotetraploid cotton (Gossypium hirsutum and Gossypium barbadense, AADD) and its extant AA (Gossypium arboreum) and DD (Gossypium raimondii) progenitors. We observed distinct DHS distributions between G. arboreum and G. raimondii. In contrast, the DHSs of the two subgenomes of G. hirsutum and G. barbadense showed a convergent distribution. This convergent distribution of DHS was also present in the wild allotetraploids Gossypium darwinii and G. hirsutum var. yucatanense, but absent from a resynthesized hybrid of G. arboreum and G. raimondii, suggesting that it may be a common feature in polyploids, and not a consequence of domestication after polyploidization. We revealed that putative cis-regulatory elements (CREs) derived from polyploidization-related DHSs were dominated by several families, including Dof, ERF48, and BPC1. Strikingly, 56.6% of polyploidization-related DHSs were derived from transposable elements (TEs). Moreover, we observed positive correlations between DHS accessibility and the histone marks H3K4me3, H3K27me3, H3K36me3, H3K27ac, and H3K9ac, indicating that coordinated interplay among histone modifications, TEs, and CREs drives the DHS landscape dynamics under polyploidization. Collectively, these findings advance our understanding of the regulatory architecture in plants and underscore the complexity of regulome evolution during polyploidization.

Evolutionary divergence of duplicated genomes in newly described allotetraploid cottons.

Allotetraploid cotton (Gossypium) species represents a model system for the study of plant polyploidy, molecular evolution, and domestication. Here, chromosome-scale genome sequences were obtained and assembled for two recently described wild species of tetraploid cotton, Gossypium ekmanianum [(AD)6, Ge] and Gossypium stephensii [(AD)7, Gs], and one early form of domesticated Gossypium hirsutum, race punctatum [(AD)1, Ghp]. Based on phylogenomic analysis, we provide a dated whole-genome level perspective for the evolution of the tetraploid Gossypium clade and resolved the evolutionary relationships of Gs, Ge, and domesticated G. hirsutum. We describe genomic structural variation that arose during Gossypium evolution and describe its correlates-including phenotypic differentiation, genetic isolation, and genetic convergence-that contributed to cotton biodiversity and cotton domestication. Presence/absence variation is prominent in causing cotton genomic structural variations. A presence/absence variation-derived gene encoding a phosphopeptide-binding protein is implicated in increasing fiber length during cotton domestication. The relatively unimproved Ghp offers the potential for gene discovery related to adaptation to environmental challenges. Expanded gene families enoyl-CoA δ isomerase 3 and RAP2-7 may have contributed to abiotic stress tolerance, possibly by targeting plant hormone-associated biochemical pathways. Our results generate a genomic context for a better understanding of cotton evolution and for agriculture.

Dynamic DNA methylation modifications in the cold stress response of cassava.

Cassava, a crucial tropical crop, faces challenges from cold stress, necessitating an exploration of its molecular response. Here, we investigated the role of DNA methylation in moderating the response to moderate cold stress (10 °C) in cassava. Using whole-genome bisulfite sequencing, we examined DNA methylation patterns in leaf blades and petioles under control conditions, 5 h, and 48 h of cold stress. Tissue-specific responses were observed, with leaf blades exhibiting subtle changes, while petioles displayed a pronounced decrease in methylation levels under cold stress. We identified cold stress-induced differentially methylated regions (DMRs) that demonstrated both tissue and treatment specificity. Importantly, these DMRs were enriched in genes with altered expression, implying functional relevance. The cold-response transcription factor ERF105 associated with DMRs emerged as a significant and conserved regulator across tissues and treatments. Furthermore, we investigated DNA methylation dynamics in transposable elements, emphasizing the sensitivity of MITEs with bHLH binding motifs to cold stress. These findings provide insights into the epigenetic regulation of response to cold stress in cassava, contributing to an understanding of the molecular mechanisms underlying stress adaptation in this tropical plant.

High-quality chromosome-level genome assembly and multi-omics analysis of rosemary (Salvia rosmarinus) reveals new insights into the environmental and genome adaptation.

High-quality genome of rosemary (Salvia rosmarinus) represents a valuable resource and tool for understanding genome evolution and environmental adaptation as well as its genetic improvement. However, the existing rosemary genome did not provide insights into the relationship between antioxidant components and environmental adaptability. In this study, by employing Nanopore sequencing and Hi-C technologies, a total of 1.17 Gb (97.96%) genome sequences were mapped to 12 chromosomes with 46 121 protein-coding genes and 1265 non-coding RNA genes. Comparative genome analysis reveals that rosemary had a closely genetic relationship with Salvia splendens and Salvia miltiorrhiza, and it diverged from them approximately 33.7 million years ago (MYA), and one whole-genome duplication occurred around 28.3 MYA in rosemary genome. Among all identified rosemary genes, 1918 gene families were expanded, 35 of which are involved in the biosynthesis of antioxidant components. These expanded gene families enhance the ability of rosemary adaptation to adverse environments. Multi-omics (integrated transcriptome and metabolome) analysis showed the tissue-specific distribution of antioxidant components related to environmental adaptation. During the drought, heat and salt stress treatments, 36 genes in the biosynthesis pathways of carnosic acid, rosmarinic acid and flavonoids were up-regulated, illustrating the important role of these antioxidant components in responding to abiotic stresses by adjusting ROS homeostasis. Moreover, cooperating with the photosynthesis, substance and energy metabolism, protein and ion balance, the collaborative system maintained cell stability and improved the ability of rosemary against harsh environment. This study provides a genomic data platform for gene discovery and precision breeding in rosemary. Our results also provide new insights into the adaptive evolution of rosemary and the contribution of antioxidant components in resistance to harsh environments.

A cell wall-localized ß-1,3-glucanase promotes fiber cell elongation and secondary cell wall deposition.

ß-1,3-glucanase functions in plant physiological and developmental processes. However, how ß-1,3-glucanase participates in cell wall development remains largely unknown. Here, we answered this question by examining the role of GhGLU18, a ß-1,3-glucanase, in cotton (Gossypium hirsutum) fibers, in which the content of ß-1,3-glucan changes dynamically from 10% of the cell wall mass at the onset of secondary wall deposition to <1% at maturation. GhGLU18 was specifically expressed in cotton fiber with higher expression in late fiber elongation and secondary cell wall (SCW) synthesis stages. GhGLU18 largely localized to the cell wall and was able to hydrolyze ß-1,3-glucan in vitro. Overexpression of GhGLU18 promoted polysaccharide accumulation, cell wall reconstruction, and cellulose synthesis, which led to increased fiber length and strength with thicker cell walls and shorter pitch of the fiber helix. However, GhGLU18-suppressed cotton resulted in opposite phenotypes. Additionally, GhGLU18 was directly activated by GhFSN1 (fiber SCW-related NAC1), a NAC transcription factor reported previously as the master regulator in SCW formation during fiber development. Our results demonstrate that cell wall-localized GhGLU18 promotes fiber elongation and SCW thickening by degrading callose and enhancing polysaccharide metabolism and cell wall synthesis.

Diversity, Variance, and Stability of Root Phenes of Peanut (Arachis hypogaea L.).

Root phenes are associated with the absorptive efficiency of water and fertilizers. However, there are few reports on the genetic variation and stability of peanut (Arachis hypogaea L.) root architecture under different environments. In this study, the diversity, variance and stability of root phenes of 89 peanut varieties were investigated with shovelomics (high throughput phenotyping of root system architecture) for two years in both field and laboratory experiments. The root phenes of these peanut genotypes presented rich diversity; for example, the value of total root length (TRL) ranged from 347.84 cm to 1013.80 cm in the field in 2018, and from 55.14 cm to 206.22 cm in the laboratory tests. The root phenes of different genotypes varied differently; for example, the coefficient of variation (CV) of TRL ranged from 24.0 to 83.5 across the two-year field test. Field and laboratory evaluations were highly correlated, especially on lateral root density (LRD) and root angle (RA), and the quadrant graph analysis of LRD and RA implied that 69.7% of the roots belong to the same type. These not only further reflect root phenes stability through different environment but also demonstrate that some root phenes identified at early stage can indicate their status at later growth stage. In addition, root phenes showed a strong correlation with shoot growth, especially root dry weight (RDW), TRL and（nodule number）NN. Thus, laboratory tests in combination with field shovelomics can efficiently screen and select genotypes with contrasting root phenes to optimize water and nutrient management.

A novel bispecific antibody drug conjugate targeting HER2 and HER3 with potent therapeutic efficacy against breast cancer.

Antibody drug conjugate (ADC) therapy has become one of the most promising approaches in cancer immunotherapy. Bispecific targeting could enhance the efficacy and safety of ADC by improving its specificity, affinity and internalization. In this study we constructed a HER2/HER3-targeting bispecific ADC (BsADC) and characterized its physiochemical properties, target specificity and internalization in vitro, and assessed its anti-tumor activities in breast cancer cell lines and in animal models. The HER2/HER3-targeting BsADC had a drug to antibody ratio (DAR) of 2.89, displayed a high selectivity against the target JIMT-1 breast cancer cells in vitro, as well as a slightly higher level of internalization than HER2- or HER3-monospecific ADCs. More importantly, the bispecific ADC potently inhibited the viability of MCF7, JIMT-1, BT474, BxPC-3 and SKOV-3 cancer cells in vitro. In JIMT-1 breast cancer xenograft mice, a single injection of bispecific ADC (3 mg/kg, i.v.) significantly inhibited the tumor growth with an efficacy comparable to that caused by combined injection of HER2 and HER3-monospecific ADCs (3 mg/kg for each). Our study demonstrates that the bispecific ADC concept can be applied to development of more potent new cancer therapeutics than the monospecific ADCs.

T cell-redirecting antibody for treatment of solid tumors via targeting mesothelin.

T cell engaging bispecific antibodies (TCBs) have recently become significant in cancer treatment. In this study we developed MSLN490, a novel TCB designed to target mesothelin (MSLN), a glycosylphosphatidylinositol (GPI)-linked glycoprotein highly expressed in various cancers, and evaluated its efficacy against solid tumors. CDR walking and phage display techniques were used to improve affinity of the parental antibody M912, resulting in a pool of antibodies with different affinities to MSLN. From this pool, various bispecific antibodies (BsAbs) were assembled. Notably, MSLN490 with its IgG-[L]-scFv structure displayed remarkable anti-tumor activity against MSLN-expressing tumors (EC50: 0.16 pM in HT-29-hMSLN cells). Furthermore, MSLN490 remained effective even in the presence of non-membrane-anchored MSLN (soluble MSLN). Moreover, the anti-tumor activity of MSLN490 was enhanced when combined with either Atezolizumab or TAA × CD28 BsAbs. Notably, a synergistic effect was observed between MSLN490 and paclitaxel, as paclitaxel disrupted the immunosuppressive microenvironment within solid tumors, enhancing immune cells infiltration and improved anti-tumor efficacy. Overall, MSLN490 exhibits robust anti-tumor activity, resilience to soluble MSLN interference, and enhanced anti-tumor effects when combined with other therapies, offering a promising future for the treatment of a variety of solid tumors. This study provides a strong foundation for further exploration of MSLN490's clinical potential.

Re-Engineering Therapeutic Anti-Aß Monoclonal Antibody to Target Amyloid Light Chain.

Amyloidosis involves the deposition of misfolded proteins. Even though it is caused by different pathogenic mechanisms, in aggregate, it shares similar features. Here, we tested and confirmed a hypothesis that an amyloid antibody can be engineered by a few mutations to target a different species. Amyloid light chain (AL) and ß-amyloid peptide (Aß) are two therapeutic targets that are implicated in amyloid light chain amyloidosis and Alzheimer's disease, respectively. Though crenezumab, an anti-Aß antibody, is currently unsuccessful, we chose it as a model to computationally design and prepare crenezumab variants, aiming to discover a novel antibody with high affinity to AL fibrils and to establish a technology platform for repurposing amyloid monoclonal antibodies. We successfully re-engineered crenezumab to bind both Aß42 oligomers and AL fibrils with high binding affinities. It is capable of reversing Aß42-oligomers-induced cytotoxicity, decreasing the formation of AL fibrils, and alleviating AL-fibrils-induced cytotoxicity in vitro. Our research demonstrated that an amyloid antibody could be engineered by a few mutations to bind new amyloid sequences, providing an efficient way to reposition a therapeutic antibody to target different amyloid diseases.

Dynamic physiological and transcriptomic changes reveal memory effects of salt stress in maize.

BACKGROUND: Pre-exposing plants to abiotic stresses can induce stress memory, which is crucial for adapting to subsequent stress exposure. Although numerous genes involved in salt stress response have been identified, the understanding of memory responses to salt stress remains limited. RESULTS: In this study, we conducted physiological and transcriptional assays on maize plants subjected to recurrent salt stress to characterize salt stress memory. During the second exposure to salt stress, the plants exhibited enhanced salt resistance, as evidenced by increased proline content and higher POD and SOD activity, along with decreased MDA content, indicative of physiological memory behavior. Transcriptional analysis revealed fewer differentially expressed genes and variations in response processes during the second exposure compared to the first, indicative of transcriptional memory behavior. A total of 2,213 salt stress memory genes (SMGs) were identified and categorized into four response patterns. The most prominent group of SMGs consisted of genes with elevated expression during the first exposure to salt stress but reduced expression after recurrent exposure to salt stress, or vice versa ([+ / -] or [- / +]), indicating that a revised response is a crucial process in plant stress memory. Furthermore, nine transcription factors (TFs) (WRKY40, WRKY46, WRKY53, WRKY18, WRKY33, WRKY70, MYB15, KNAT7, and WRKY54) were identified as crucial factors related to salt stress memory. These TFs regulate over 53% of SMGs, underscoring their potential significance in salt stress memory. CONCLUSIONS: Our study demonstrates that maize can develop salt stress memory, and the genes identified here will aid in the genetic improvement of maize and other crops.

scRNASequest: an ecosystem of scRNA-seq analysis, visualization, and publishing.

BACKGROUND: Single-cell RNA sequencing is a state-of-the-art technology to understand gene expression in complex tissues. With the growing amount of data being generated, the standardization and automation of data analysis are critical to generating hypotheses and discovering biological insights. RESULTS: Here, we present scRNASequest, a semi-automated single-cell RNA-seq (scRNA-seq) data analysis workflow which allows (1) preprocessing from raw UMI count data, (2) harmonization by one or multiple methods, (3) reference-dataset-based cell type label transfer and embedding projection, (4) multi-sample, multi-condition single-cell level differential gene expression analysis, and (5) seamless integration with cellxgene VIP for visualization and with CellDepot for data hosting and sharing by generating compatible h5ad files. CONCLUSIONS: We developed scRNASequest, an end-to-end pipeline for single-cell RNA-seq data analysis, visualization, and publishing. The source code under MIT open-source license is provided at https://github.com/interactivereport/scRNASequest . We also prepared a bookdown tutorial for the installation and detailed usage of the pipeline: https://interactivereport.github.io/scRNAsequest/tutorial/docs/ . Users have the option to run it on a local computer with a Linux/Unix system including MacOS, or interact with SGE/Slurm schedulers on high-performance computing (HPC) clusters.

Virus-Based CRISPR/Cas9 Genome Editing in Plants.

CRISPR/Cas9 is a versatile tool for plant gene function studies and crop improvement. However, traditional CRISPR/Cas9-mediated genome editing requires plant tissue culture that is both time-consuming and genotype-dependent. Ma et al. recently reported a novel virus-based method for delivering CRISPR/Cas9 into plant cells, and this should further expand the application of CRISPR/Cas9-mediated genome editing.

SpRY: Engineered CRISPR/Cas9 Harnesses New Genome-Editing Power.

Due to protospacer adjacent motif (PAM) requirements, CRISPR/Cas9 cannot access many genetic loci. A recent study by Walton et al. structurally engineered Streptococcus pyogenes Cas9 (SpCas9) to near-PAMless SpRY that can target most DNA sequences with high editing efficiency and flexibility. This newly engineered SpRY will potentially expand genome-editing capabilities for basic and applied research.

A review on CRISPR/Cas: a versatile tool for cancer screening, diagnosis, and clinic treatment.

Cancer is one of the leading causes of death worldwide and it has the trend of increase incidence. However, in the past decades, as quickly developed new technologies and modified old techniques for cancer screening, diagnosis, and treatment, the cancer-caused mortality rates dropped quickly, and the survival times of cancer patients are enhanced. However, the current death rate is still about 50% and the survival patients always suffer from the side effect of current cancer treatments. Recently developed Nobel Prize-winning CRISPR/Cas technology provides new hope on cancer screening, early diagnosis, and clinic treatment as well as new drug development. Currently, four major CRISPR/Cas9-derived genome editors, CRISPR/Cas9 nucleotide sequence editor, CRISPR/Cas base editor (BE), CRISPR prime editor (PE), and CRISPR interference (CRISPRi) (including both CRISPRa and CRISPRr), were well developed and used to various research and applications, including cancer biology study and cancer screening, diagnosis, and treatment. Additionally, CRISPR/Cas12 and CRISPR/Cas13 genome editors were also widely used in cancer-related basic and applied research as well as treatment. Cancer-associated SNPs and genetic mutations as well as both oncogenes and tumor suppressor genes are perfect targets for CRISPR/Cas-based gene therapy for cancer treatment. CRISPR/Cas is also employed to modify and generate new Chimeric antigen receptor (CAR) T-cells for improving its safety, efficiency, and longer-time last for treating various cancers. Currently, there are many clinic trails of CRISPR-based gene therapy for cancer treatments. Although all CRISPR/Cas-derived genome and epigenome tools are promising methods for cancer biology study and treatment, the efficiency and long term-safety are still the major concerns for CRISPR-based gene therapy. Developing new CRISPR/Cas delivery methods and reducing the potential side effects, including off-target impacts, will enhance CRISPR/Cas application in cancer-related research, diagnosis, and therapeutical treatment.

RefFinder: a web-based tool for comprehensively analyzing and identifying reference genes.

Although many genes may serve as reference genes, they may cause different expression patterns by selecting different reference genes because no single gene is expressed consistently in all tested tissues of an organism under all environmental and developmental conditions. Thus, it is becoming increasingly important and necessary to identify suitable reference genes before performing gene expression analysis. Currently, there are several computational tools available for evaluating the stability of candidate reference genes. These tools are based on different statistical algorithms and may produce different rankings in stability within the same reference gene study. To date, the RefFinder is the only web-based tool available for comparing and evaluating housekeeping genes as candidates to be reference genes. In this tool, we integrated the four currently available computational programs (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) into a web-based tool for evaluating the stability and reliability of reference genes. According to the gene stability rankings derived from the four programs, we assigned an appropriate weight to each gene and calculated the geometric mean of weights for the final rankings. Aside from the overall ranking, a single program or combination of the four programs can be selected for evaluating the ranking of candidate reference genes. This tool has been widely used and validated by many research laboratories around the world. You may use this tool at http://www.heartcure.com.au/reffinder/  or  https://blooge.cn/RefFinder/ . You can also download this algorithm program from https://github.com/fulxie/RefFinder and setup on your own computer. RefFinder is developed by PHP. Users can deploy it to a Php-based server (Apache + PHP) and run it.

The resilient cotton plant: uncovering the effects of stresses on secondary metabolomics and its underlying molecular mechanisms.

Cotton is an important fiber crop cultivated around the world under diverse climate conditions and generates billions of dollars in annual revenue globally. Biotic and abiotic stresses have caused reduction in yield and productivity of cotton crops. In this review, we comprehensively analyzed and summarized the effect of biotic and abiotic stress on secondary metabolite production in cotton. The development of cotton varieties with improved tolerance against abiotic and biotic stress can play an important role in sustainable cotton production. Under stress conditions, plants develop a variety of defense mechanisms such as initiating signaling functions to upregulate defense responsive genes and accumulation of secondary metabolites. Understanding the impact of stress on secondary metabolite production in cotton is crucial for developing strategies to alleviate the negative effects of stress on crop yield and quality. Further, the potential industrial applications of these secondary metabolites in cotton, such as gossypol, could provide new opportunities for sustainable cotton production and the development of value-added products. Additionally, transgenic and genome-edited cotton cultivars can be developed to provide tolerance to both abiotic and biotic stress in cotton production.

Insights to Gossypium defense response against Verticillium dahliae: the Cotton Cancer.

The soil-borne pathogen Verticillium dahliae, also referred as "The Cotton Cancer," is responsible for causing Verticillium wilt in cotton crops, a destructive disease with a global impact. To infect cotton plants, the pathogen employs multiple virulence mechanisms such as releasing enzymes that degrade cell walls, activating genes that contribute to virulence, and using protein effectors. Conversely, cotton plants have developed numerous defense mechanisms to combat the impact of V. dahliae. These include strengthening the cell wall by producing lignin and depositing callose, discharging reactive oxygen species, and amassing hormones related to defense. Despite the efforts to develop resistant cultivars, there is still no permanent solution to Verticillium wilt due to a limited understanding of the underlying molecular mechanisms that drive both resistance and pathogenesis is currently prevalent. To address this challenge, cutting-edge technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), host-induced gene silencing (HIGS), and gene delivery via nano-carriers could be employed as effective alternatives to control the disease. This article intends to present an overview of V. dahliae virulence mechanisms and discuss the different cotton defense mechanisms against Verticillium wilt, including morphophysiological and biochemical responses and signaling pathways including jasmonic acid (JA), salicylic acid (SA), ethylene (ET), and strigolactones (SLs). Additionally, the article highlights the significance of microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) in gene expression regulation, as well as the different methods employed to identify and functionally validate genes to achieve resistance against this disease. Gaining a more profound understanding of these mechanisms could potentially result in the creation of more efficient strategies for combating Verticillium wilt in cotton crops.

Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus.

BACKGROUND: Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species. METHODS AND RESULTS: The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV. CONCLUSIONS: CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

Long-term passaging of pseudo-typed SARS-CoV-2 reveals the breadth of monoclonal and bispecific antibody cocktails.

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses challenges to the effectiveness of neutralizing antibodies. Rational design of antibody cocktails is a realizable approach addressing viral immune evasion. However, evaluating the breadth of antibody cocktails is essential for understanding the development potential. Here, based on a replication competent vesicular stomatitis virus model that incorporates the spike of SARS-CoV-2 (VSV-SARS-CoV-2), we evaluated the breadth of a number of antibody cocktails consisting of monoclonal antibodies and bispecific antibodies by long-term passaging the virus in the presence of the cocktails. Results from over two-month passaging of the virus showed that 9E12 + 10D4 + 2G1 and 7B9-9D11 + 2G1 from these cocktails were highly resistant to random mutation, and there was no breakthrough after 30 rounds of passaging. As a control, antibody REGN10933 was broken through in the third passage. Next generation sequencing was performed and several critical mutations related to viral evasion were identified. These mutations caused a decrease in neutralization efficiency, but the reduced replication rate and ACE2 susceptibility of the mutant virus suggested that they might not have the potential to become epidemic strains. The 9E12 + 10D4 + 2G1 and 7B9-9D11 + 2G1 cocktails that picked from the VSV-SARS-CoV-2 system efficiently neutralized all current variants of concern and variants of interest including the most recent variants Delta and Omicron, as well as SARS-CoV-1. Our results highlight the feasibility of using the VSV-SARS-CoV-2 system to develop SARS-CoV-2 antibody cocktails and provide a reference for the clinical selection of therapeutic strategies to address the mutational escape of SARS-CoV-2.