Search

Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

Viral infectious diseases are a devastating and continuing threat to human and animal health. Receptor binding is the key step for viral entry into host cells. Therefore, recognizing viral receptors is fundamental for understanding the potential tissue tropism or host range of these pathogens. The rapid advancement of single-cell RNA sequencing (scRNA-seq) technology has paved the way for studying the expression of viral receptors in different tissues of animal species at single-cell resolution, resulting in huge scRNA-seq datasets. However, effectively integrating or sharing these datasets among the research community is challenging, especially for laboratory scientists. In this study, we manually curated up-to-date datasets generated in animal scRNA-seq studies, analyzed them using a unified processing pipeline, and comprehensively annotated 107 viral receptors in 142 viruses and obtained accurate expression signatures in 2 100 962 cells from 47 animal species. Thus, the VThunter database provides a user-friendly interface for the research community to explore the expression signatures of viral receptors. VThunter offers an informative and convenient resource for scientists to better understand the interactions between viral receptors and animal viruses and to assess viral pathogenesis and transmission in species. Database URL: https://db.cngb.org/VThunter/.

The brain of the domestic pig (Sus scrofa domesticus) has drawn considerable attention due to its high similarities to that of humans. However, the cellular compositions of the pig brain (PB) remain elusive. Here we investigated the single-nucleus transcriptomic profiles of five regions of the PB (frontal lobe, parietal lobe, temporal lobe, occipital lobe, and hypothalamus) and identified 21 cell subpopulations. The cross-species comparison of mouse and pig hypothalamus revealed the shared and specific gene expression patterns at the single-cell resolution. Furthermore, we identified cell types and molecular pathways closely associated with neurological disorders, bridging the gap between gene mutations and pathogenesis. We reported, to our knowledge, the first single-cell atlas of domestic pig cerebral cortex and hypothalamus combined with a comprehensive analysis across species, providing extensive resources for future research regarding neural science, evolutionary developmental biology, and regenerative medicine.

Olfaction, the sense of smell, is a fundamental trait crucial to many species. The olfactory bulb (OB) plays pivotal roles in processing and transmitting odor information from the environment to the brain. The cellular heterogeneity of the mouse OB has been studied using single-cell RNA sequencing. However, the epigenetic landscape of the mOB remains mostly unexplored. Herein, we apply single-cell assay for transposase-accessible chromatin sequencing to profile the genome-wide chromatin accessibility of 9,549 single cells from the mOB. Based on single-cell epigenetic signatures, mOB cells are classified into 21 clusters corresponding to 11 cell types. We identify distinct sets of putative regulatory elements specific to each cell cluster from which putative target genes and enriched potential functions are inferred. In addition, the transcription factor motifs enriched in each cell cluster are determined to indicate the developmental fate of each cell lineage. Our study provides a valuable epigenetic data set for the mOB at single-cell resolution, and the results can enhance our understanding of regulatory circuits and the therapeutic capacity of the OB at the single-cell level.

Horseshoe bats (Rhinolophus sinicus) might help maintain coronaviruses severely affecting human health, such as SARS-CoV and SARS-CoV-2. It has long been suggested that bats may be more tolerant of viral infection than other mammals due to their unique immune system, but the exact mechanism remains to be fully explored. During the COVID-19 pandemic, multiple animal species were diseased by SARS-CoV-2 infection, especially in the respiratory system. Herein, single-cell transcriptomic data of the lungs of a horseshoe bat, a cat, a tiger, and a pangolin were generated. The receptor distribution of twenty-eight respiratory viruses belonging to fourteen viral families were characterized for the four species. Comparison on the immune-related transcripts further revealed limited cytokine activations in bats, which might explain the reason why bats experienced only mild diseases or even no symptoms upon virus infection. Our findings might increase our understanding of the immune background of horseshoe bats and their insensitivity to virus infections.

Civets are small mammals belonging to the family Viverridae. The masked palm civets (Paguma larvata) served as an intermediate host in the bat-to-human transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) in 20031. Because of their unique role in the SARS outbreak, civets were suspected as a potential intermediate host of SARS-CoV-2, the etiological pathogen of the COVID-19 pandemic. Besides their susceptibility to coronaviruses, civets can also be infected by other viruses, such as canine distemper viruses2, parvoviruses3, influenza viruses4, etc. Regarding the ecological and economical role of civets, it is vital to evaluate the potential threats from different pathogens to these animals. Receptor binding is a necessary step for virus entry into host cells. Understanding the distribution of receptors of various viruses provides hints to their potential tissue tropisms. Herein, we characterized the cell atlas of five important organs (the frontal lobe, lung, liver, spleen and kidney) of masked palm civets (Paguma larvata) and described the expression profiles of receptor associated genes of 132 viruses from 25 families, including 16 viruses from 10 families reported before that can attack civets and 116 viruses with little infection record.

BackgroundImmune cells play important roles in mediating immune response and host defense against invading pathogens. However, insights into the molecular mechanisms governing circulating immune cell diversity among multiple species are limited.MethodsIn this study, we compared the single-cell transcriptomes of 77□957 immune cells from 12 species using single-cell RNA-sequencing (scRNA-seq). Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells.ResultsResults revealed the heterogeneity and compositions of circulating immune cells among 12 different species. Additionally, we explored the conserved and divergent cellular crosstalk and genetic regulatory networks among vertebrate immune cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells.ConclusionsThis study is the first to provide a comprehensive analysis of the cross-species single-cell atlas for peripheral blood mononuclear cells (PBMCs). This research should advance our understanding of the cellular taxonomy and fundamental functions of PBMCs, with important implications in evolutionary biology, developmental biology, and immune system disorders.

Vertebrate evolution was accompanied by two rounds of whole-genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single-cell sequencing has been widely used to construct the developmental cell atlas of several representative species of vertebrates (human, mouse, zebrafish, and frog) and tunicates (sea squirts). Here, we perform single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated, we constructed a developmental tree for amphioxus cell fate commitment and lineage specification and characterize the underlying key regulators and genetic regulatory networks. The data are publicly available on the online platform AmphioxusAtlas.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) issued a significant and urgent threat to global health. The exact animal origin of SARS-CoV-2 remains obscure and understanding its host range is vital for preventing interspecies transmission. Previously, we have assessed the target cell profiles of SARS-CoV-2 in pets, livestock, poultry and wild animals. Herein, we expand this investigation to a wider range of animal species and viruses to provide a comprehensive source for large-scale screening of potential virus hosts. Single cell atlas for several mammalian species (alpaca, hamster, hedgehog, chinchilla etc.), as well as comparative atlas for lung, brain and peripheral blood mononuclear cells (PBMC) for various lineages of animals were constructed, from which we systemically analyzed the virus entry factors for 113 viruses over 20 species from mammalians, birds, reptiles, amphibians and invertebrates. Conserved cellular connectomes and regulomes were also identified, revealing the fundamental cell-cell and gene-gene cross-talks between these species. Overall, our study could help identify the potential host range and tissue tropism of SARS-CoV-2 and a diverse set of viruses and reveal the host-virus co-evolution footprints.

Vertebrate evolution was accompanied with two rounds of whole genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single cell sequencing has been widely employed to construct the developmental cell atlas of several key species of vertebrates (human, mouse, zebrafish and frog) and tunicate (sea squirts). Here, we performed single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated we constructed the developmental tree for amphioxus cell fate commitment and lineage specification, and revealed the underlying key regulators and genetic regulatory networks. The generated data were integrated into an online platform, AmphioxusAtlas, for public access at http://120.79.46.200:81/AmphioxusAtlas.

we established the broadest single-nucleus RNA sequencing study to date, covering 11 representative species, including pets (cat, dog, hamster, and lizard), livestock (goat and rabbit), poultry (duck and pigeon), and wildlife (pangolin, tiger, and deer).

SummarySystem-wide molecular characteristics of COVID-19, especially in those patients without comorbidities, have not been fully investigated. We compared extensive molecular profiles of blood samples from 231 COVID-19 patients, ranging from asymptomatic to critically ill, importantly excluding those with any comorbidities. Amongst the major findings, asymptomatic patients were characterized by highly activated anti-virus interferon, T/natural killer (NK) cell activation, and transcriptional upregulation of inflammatory cytokine mRNAs. However, given very abundant RNA binding proteins (RBPs), these cytokine mRNAs could be effectively destabilized hence preserving normal cytokine levels. In contrast, in critically ill patients, cytokine storm due to RBPs inhibition and tryptophan metabolites accumulation contributed to T/NK cell dysfunction. A machine-learning model was constructed which accurately stratified the COVID-19 severities based on their multi-omics features. Overall, our analysis provides insights into COVID-19 pathogenesis and identifies targets for intervening in treatment.

A few animals have been suspected to be intermediate hosts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a large-scale single-cell screening of SARS-CoV-2 target cells on a wide variety of animals is missing. Here, we constructed the single-cell atlas for 11 representative species in pets, livestock, poultry, and wildlife. Notably, the proportion of SARS-CoV-2 target cells in cat was found considerably higher than other species we investigated and SARS-CoV-2 target cells were detected in multiple cell types of domestic pig, implying the necessity to carefully evaluate the risk of cats during the current COVID-19 pandemic and keep pigs under surveillance for the possibility of becoming intermediate hosts in future coronavirus outbreak. Furthermore, we screened the expression patterns of receptors for 144 viruses, resulting in a comprehensive atlas of virus target cells. Taken together, our work provides a novel and fundamental strategy to screen virus target cells and susceptible species, based on single-cell transcriptomes we generated for domesticated animals and wildlife, which could function as a valuable resource for controlling current pandemics and serve as an early warning system for coping with future infectious disease threats.

Down syndrome (DS) is one of the most common human birth defects caused by trisomy 21 (T21), leading to a variety of cognitive impairments. The cellular composition of human brain has been explored using single cell RNA sequencing in both physiological and pathological conditions. However, the cellular heterogeneity of human brain with chromosome aneuploidy is largely unknown. Here, we profiled the transcriptome of 36046 cells in cerebral cortex of T21 human fetus, covering frontal lobe, parietal lobe, occipital lobe and temporal lobe. Intriguingly, we detected several genes positively associated with neurons maturation was dysregulated in T21 frontal cortex (HIC2, POU2F2, ZGLP1 and FOXK1). To share, explore and utilized the data resources of T21 cerebral cortex, we developed a comprehensive platform named T21atlas, composing of two functional modules (T21cluster and T21talk). Overall, our study provides, as far as we know, the first single cell atlas for T21 cerebral cortex, which could promote our understanding of the molecular mechanism of DS at an unprecedented resolution and could potentially facilitate the development of novel clinical therapeutics against T21.

Infrared (IR) thermography is applied to study the thermal behavior of laser metal-wire additive manufacturing (AM) in vacuum. IR thermal images are obtained and analyzed. Based upon IR thermography analysis, the thermal cycle, remelting and cooling rate in AM process is discussed. In addition, the width of cladding layer is predicted and defect like lack of fusion is detected.

Domestic pig (Sus scrofa domesticus) has drawn much attention from researchers worldwide due to its implications in evolutionary biology, regenerative medicine and agriculture. The brain atlas of Homo sapiens (primate), Mus musculus (rodent), Danio rerio (fish) and Drosophila melanogaster (insect) have been constructed at single cell resolution, however, the cellular compositions of pig brain remain largely unexplored. In this study, we investigated the single-cell transcriptomic profiles of five distinct regions of domestic pig brain, from which we identified 21 clusters corresponding to six major cell types, characterized by unique spectrum of gene expression. By spatial comparison, we identified cell types enriched or depleted in certain brain regions. Inter-species comparison revealed cell-type similarities and divergences in hypothalamus between mouse and pig, providing invaluable resources for the evolutionary exploration of brain functions at single cell level. Besides, our study revealed cell types and molecular pathways closely associated with several diseases (obesity, anorexia, bulimia, epilepsy, intellectual disability, and autism spectrum disorder), bridging the gap between gene mutations and pathological phenotypes, which might be of great use to the development precise therapies against neural system disorders. Taken together, we reported, so far as we know, the first single cell brain atlas of Sus scrofa domesticus, followed by comprehensive comparisons across brain region and species, which could throw light upon future evo-devo, regenerative medicine, and agricultural studies.

Retina, located in the innermost layer of the eye of human, holds the decisive role in visual perception. Dissecting the heterogeneity of retina is essential for understanding the mechanism of vision formation and even the development of central nervous system (CNS). Here, we performed single cell RNA-seq, analyzed 57,832 cells from human infant donors, resulting in 20 distinct clusters representing major cell types in retina: rod photoreceptors, cone photoreceptors, bipolar cells, horizontal cells, amacrine cells, Muller glia cells and microglia. We next constructed extensive networks of intercellular communication and identified ligand-receptor interactions playing crucial roles in regulating neural cell development and immune homeostasis in retina. Though re-clustering, we identified known subtypes in cone PRs and additional unreported subpopulations and corresponding markers in rod PRs as well as bipolar cells. Additionally, we linked inherited retinal disease to certain cell subtypes or subpopulations through enrichment analysis. Intriguingly, we found that status and functions of photoreceptors changed drastically between early and late retina. Overall, our study offers the first retinal cell atlas in human infants, dissecting the heterogeneity of retina and identifying the key molecules in the developmental process, which provides an important resource that will pave the way for retina development mechanism research and regenerative medicine concerning retinal biology.

Retina is a crucial tissue for the capturing and processing of light stimulus. Characterization of the retina at single cell level is essential for the understanding of its biological functions. A variety of abnormalities in terms of morphology and function were reported in T21 retina. To evaluate the effects of chromosome aneuploidy on retina development, we characterized single cell transcriptional profiles of a T21 fetus and performed comprehensive bioinformatic analyses. Our data revealed the diversity and heterogeneity of cellular compositions in T21 retina. In total, we identified seven major cell types, and detected several subtypes within each cell type, followed by the detection of corresponding molecular markers including previously reported ones and a series of novel markers. Our analyses identified extensive communication networks between distinct cellular types, among which a few ligand-receptor interactions were associated with the development of retina and immunoregulatory interactions. Taken together, our data provided the first single cell transcriptome profile for human T21 retina which facilitates our understanding on the dosage effects of chromosome 21 on the development of retina.

BackgroundInvestigating cell fate decision and subpopulation specification in the context of the neural lineage is fundamental to understanding neurogenesis and neurodegenerative diseases. The differentiation process of neural-tube-like rosettesin vitrois representative of neural tube structures, which are composed of radially organized, columnar epithelial cells and give rise to functional neural cells. However, the underlying regulatory network of cell fate commitment during early neural differentiation remains elusive.ResultsIn this study, we investigated the genome-wide transcriptome profile of single cells from six consecutive reprogramming and neural differentiation time points and identified cellular subpopulations present at each differentiation stage. Based on the inferred reconstructed trajectory and the characteristics of subpopulations contributing the most towards commitment to the central nervous system (CNS) lineage at each stage during differentiation, we identified putative novel transcription factors in regulating neural differentiation. In addition, we dissected the dynamics of chromatin accessibility at the neural differentiation stages and revealed active c/s-regulatory elements for transcription factors known to have a key role in neural differentiation as well as for those that we suggest are also involved. Further, communication network analysis demonstrated that cellular interactions most frequently occurred among embryoid body (EB) stage and each cell subpopulation possessed a distinctive spectrum of ligands and receptors associated with neural differentiation which could reflect the identity of each subpopulation.ConclusionsOur study provides a comprehensive and integrative study of the transcriptomics and epigenetics of human early neural differentiation, which paves the way for a deeper understanding of the regulatory mechanisms driving the differentiation of the neural lineage.