Discovery of deaminase functions by structure-based protein clustering.

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.

Single-Cell Transcriptome Atlas of Murine Endothelial Cells.

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

Human RTEL1 associates with Poldip3 to facilitate responses to replication stress and R-loop resolution.

RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.

Extrachromosomal circular DNA in cancer: history, current knowledge, and methods.

Extrachromosomal circular DNA (eccDNA) is a closed-circle, nuclear, nonplasmid DNA molecule found in all tested eukaryotes. eccDNA plays important roles in cancer pathogenesis, evolution of tumor heterogeneity, and therapeutic resistance. It is known under many names, including very large cancer-specific circular extrachromosomal DNA (ecDNA), which carries oncogenes and is often amplified in cancer cells. Our understanding of eccDNA has historically been limited and fragmented. To provide better a context of new and previous research on eccDNA, in this review we give an overview of the various names given to eccDNA at different times. We describe the different mechanisms for formation of eccDNA and the methods used to study eccDNA thus far. Finally, we explore the potential clinical value of eccDNA.

AMBRA1 phosphorylation by CDK1 and PLK1 regulates mitotic spindle orientation.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.

Single-nucleus transcriptome inventory of giant panda reveals cellular basis for fitness optimization under low metabolism.

BACKGROUND: Energy homeostasis is essential for the adaptation of animals to their environment and some wild animals keep low metabolism adaptive to their low-nutrient dietary supply. Giant panda is such a typical low-metabolic mammal exhibiting species specialization of extremely low daily energy expenditure. It has low levels of basal metabolic rate, thyroid hormone, and physical activities, whereas the cellular bases of its low metabolic adaptation remain rarely explored. RESULTS: In this study, we generate a single-nucleus transcriptome atlas of 21 organs/tissues from a female giant panda. We focused on the central metabolic organ (liver) and dissected cellular metabolic status by cross-species comparison. Adaptive expression mode (i.e., AMPK related) was prominently displayed in the hepatocyte of giant panda. In the highest energy-consuming organ, the heart, we found a possibly optimized utilization of fatty acid. Detailed cell subtype annotation of endothelial cells showed the uterine-specific deficiency of blood vascular subclasses, indicating a potential adaptation for a low reproductive energy expenditure. CONCLUSIONS: Our findings shed light on the possible cellular basis and transcriptomic regulatory clues for the low metabolism in giant pandas and helped to understand physiological adaptation response to nutrient stress.

Resistance to vincristine in DLBCL by disruption of p53-induced cell cycle arrest and apoptosis mediated by KIF18B and USP28.

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous Phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. The inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 of which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response suggesting that resistance to vincristine has origin in the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.

A network-based algorithm for the identification of moonlighting noncoding RNAs and its application in sepsis.

Moonlighting proteins provide more options for cells to execute multiple functions without increasing the genome and transcriptome complexity. Although there have long been calls for computational methods for the prediction of moonlighting proteins, no method has been designed for determining moonlighting long noncoding ribonucleicacidz (RNAs) (mlncRNAs). Previously, we developed an algorithm MoonFinder for the identification of mlncRNAs at the genome level based on the functional annotation and interactome data of lncRNAs and proteins. Here, we update MoonFinder to MoonFinder v2.0 by providing an extensive framework for the detection of protein modules and the establishment of RNA-module associations in human. A novel measure, moonlighting coefficient, was also proposed to assess the confidence of an ncRNA acting in a moonlighting manner. Moreover, we explored the expression characteristics of mlncRNAs in sepsis, in which we found that mlncRNAs tend to be upregulated and differentially expressed. Interestingly, the mlncRNAs are mutually exclusive in terms of coexpression when compared to the other lncRNAs. Overall, MoonFinder v2.0 is dedicated to the prediction of human mlncRNAs and thus bears great promise to serve as a valuable R package for worldwide research communities (https://cran.r-project.org/web/packages/MoonFinder/index.html). Also, our analyses provide the first attempt to characterize mlncRNA expression and coexpression properties in adult sepsis patients, which will facilitate the understanding of the interaction and expression patterns of mlncRNAs.

Genome-wide annotation of protein-coding genes in pig.

BACKGROUND: There is a need for functional genome-wide annotation of the protein-coding genes to get a deeper understanding of mammalian biology. Here, a new annotation strategy is introduced based on dimensionality reduction and density-based clustering of whole-body co-expression patterns. This strategy has been used to explore the gene expression landscape in pig, and we present a whole-body map of all protein-coding genes in all major pig tissues and organs. RESULTS: An open-access pig expression map ( www.rnaatlas.org ) is presented based on the expression of 350 samples across 98 well-defined pig tissues divided into 44 tissue groups. A new UMAP-based classification scheme is introduced, in which all protein-coding genes are stratified into tissue expression clusters based on body-wide expression profiles. The distribution and tissue specificity of all 22,342 protein-coding pig genes are presented. CONCLUSIONS: Here, we present a new genome-wide annotation strategy based on dimensionality reduction and density-based clustering. A genome-wide resource of the transcriptome map across all major tissues and organs in pig is presented, and the data is available as an open-access resource ( www.rnaatlas.org ), including a comparison to the expression of human orthologs.

Haplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing.

Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.

Systematical analysis reveals a strong cancer relevance of CREB1-regulated genes.

The transcription factor cyclic-AMP response element-binding protein 1 (CREB1) responds to cAMP level and controls the expression of target genes, which regulates nutrition partitioning. The promoters of CREB1-targeted genes responsive to cAMP have been extensively investigated and characterized with the presence of both cAMP response element and TATA box. Compelling evidence demonstrates that CREB1 also plays an essential role in promoting tumor development. However, only very few genes required for cell survival, proliferation and migration are known to be constitutively regulated by CREB1 in tumors. Their promoters mostly do not harbor any cAMP response element. Thus, it is very likely that CREB1 regulates the expressions of distinct sets of target genes in normal tissues and tumors. The whole gene network constitutively regulated by CREB1 in tumors has remained unrevealed. Here, we employ a systematical and integrative approach to decipher this gene network in the context of both tissue cultured cancer cells and patient samples. We combine transcriptomic, Rank-Rank Hypergeometric Overlap, and Chipseq analysis, to define and characterize CREB1-regulated genes in a multidimensional fashion. A strong cancer relevance of those top-ranked targets, which meet the most stringent criteria, is eventually verified by overall survival analysis of cancer patients. These findings strongly suggest the importance of genes constitutively regulated by CREB1 for their implicative involvement in promoting tumorigenesis.

Double-strand breaks in ribosomal RNA genes activate a distinct signaling and chromatin response to facilitate nucleolar restructuring and repair.

The nucleolus is a nuclear sub-domain containing the most highly transcribed genes in the genome. Hundreds of human ribosomal RNA (rRNA) genes, located in the nucleolus, rely on constant maintenance. DNA double-strand breaks (DSBs) in rRNA genes activate the ATM kinase, repress rRNA transcription and induce nucleolar cap formation. Yet how ribosomal-DNA (rDNA) lesions are detected and processed remains elusive. Here, we use CRISPR/Cas9-mediated induction of DSBs and report a chromatin response unique to rDNA depending on ATM-phosphorylation of the nucleolar protein TCOF1 and recruitment of the MRE11-RAD50-NBS1 (MRN) complex via the NBS1-subunit. NBS1- and MRE11-depleted cells fail to suppress rRNA transcription and to translocate rDNA into nucleolar caps. Furthermore, the DNA damage response (DDR) kinase ATR operates downstream of the ATM-TCOF1-MRN interplay and is required to fully suppress rRNA transcription and complete DSB-induced nucleolar restructuring. Unexpectedly, we find that DSBs in rDNA neither activate checkpoint kinases CHK1/CHK2 nor halt cell-cycle progression, yet the nucleolar-DDR protects against genomic aberrations and cell death. Our data highlight the concept of a specialized nucleolar DNA damage response (n-DDR) with a distinct protein composition, spatial organization and checkpoint communication. The n-DDR maintains integrity of ribosomal RNA genes, with implications for cell physiology and disease.

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation.

BACKGROUND: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. METHODS: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. RESULTS: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. CONCLUSIONS: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

Identification and functional characterization of mutations in LPL gene causing severe hypertriglyceridaemia and acute pancreatitis.

Hypertriglyceridaemia is a very rare disorder caused by the mutations of LPL gene, with an autosomal recessive mode of inheritance. Here, we identified two unrelated Chinese patients manifested with severe hypertriglyceridaemia and acute pancreatitis. The clinical symptoms of proband 1 are more severe than proband 2. Whole exome sequencing and Sanger sequencing were performed. Functional analysis of the identified mutations has been done. Whole exome sequencing identified two pairs of variants in LPL gene in the proband 1 (c.162C>A and c.1322+1G>A) and proband 2 (c.835C>G and c.1322+1G>A). The substitution (c.162C>A) leads to the formation of a truncated (p.Cys54*) LPL protein. The substitution (c.835C>G) leads to the replacement of leucine to valine (p.Leu279Val). The splice donor site mutation (c.1322+1G>A) leads to the formation of alternative transcripts with the loss of 134 bp in exon 8 of the LPL gene. The proband 1 and his younger son also harbouring a heterozygous variant (c.553G>T; p.Gly185Cys) in APOA5 gene. The relative expression level of the mutated LPL mRNA (c.162C>A, c.835C>G and c.1322+1G>A) showed significant differences compared to wild-type LPL mRNA, suggesting that all these three mutations affect the transcription of LPL mRNA. These three mutations (c.162C>A, c.835C>G and c.1322+1G>A) showed noticeably decreased LPL activity in cell culture medium but not in cell lysates. Here, we identified three mutations in LPL gene which causes severe hypertriglyceridaemia with acute pancreatitis in Chinese patients. We also described the significance of whole exome sequencing for identifying the candidate gene and disease-causing mutation in patients with severe hypertriglyceridaemia and acute pancreatitis.

An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools.

The CRISPR-Cas9 system has become the most promising and versatile tool for genetic manipulation applications. Albeit the technology has been broadly adopted by both academic and pharmaceutic societies, the activity (on-target) and specificity (off-target) of CRISPR-Cas9 are decisive factors for any application of the technology. Several in silico gRNA activity and specificity predicting models and web tools have been developed, making it much more convenient and precise for conducting CRISPR gene editing studies. In this review, we present an overview and comparative analysis of machine and deep learning (MDL)-based algorithms, which are believed to be the most effective and reliable methods for the prediction of CRISPR gRNA on- and off-target activities. As an increasing number of sequence features and characteristics are discovered and are incorporated into the MDL models, the prediction outcome is getting closer to experimental observations. We also introduced the basic principle of CRISPR activity and specificity and summarized the challenges they faced, aiming to facilitate the CRISPR communities to develop more accurate models for applying.

LION: a simple and rapid method to achieve CRISPR gene editing.

The RNA-guided CRISPR-Cas9 technology has paved the way for rapid and cost-effective gene editing. However, there is still a great need for effective methods for rapid generation and validation of CRISPR/Cas9 gRNAs. Previously, we have demonstrated that highly efficient generation of multiplexed CRISPR guide RNA (gRNA) expression array can be achieved with Golden Gate Assembly (GGA). Here, we present an optimized and rapid method for generation and validation in less than 1 day of CRISPR gene targeting vectors. The method (LION) is based on ligation of double-stranded gRNA oligos into CRISPR vectors with GGA followed by nucleic acid purification. Using a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay and a traffic light reporter assay, we proved that the LION-based generation of CRISPR vectors are functionally active, and equivalent to CRISPR plasmids generated by traditional methods. We also tested the activity of LION CRISPR vectors in different human cell types. The LION method presented here advances the rapid functional validation and application of CRISPR system for gene editing and simplified the CRISPR gene-editing procedures.

CRISPR-C: circularization of genes and chromosome by CRISPR in human cells.

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.

Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells.

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole-transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency-related gene network of the LIF- versus FGF-derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC-like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC-like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extent, in the piPSC-like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line-transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. 84: 229-245, 2017. © 2016 Wiley Periodicals, Inc.

Genetically modified pig models for neurodegenerative disorders.

Increasing incidence of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease has become one of the most challenging health issues in ageing humans. One approach to combat this is to generate genetically modified animal models of neurodegenerative disorders for studying pathogenesis, prognosis, diagnosis, treatment, and prevention. Owing to the genetic, anatomic, physiologic, pathologic, and neurologic similarities between pigs and humans, genetically modified pig models of neurodegenerative disorders have been attractive large animal models to bridge the gap of preclinical investigations between rodents and humans. In this review, we provide a neuroanatomical overview in pigs and summarize and discuss the generation of genetically modified pig models of neurodegenerative disorders including Alzheimer's diseases, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, and ataxia-telangiectasia. We also highlight how non-invasive bioimaging technologies such as positron emission tomography (PET), computer tomography (CT), and magnetic resonance imaging (MRI), and behavioural testing have been applied to characterize neurodegenerative pig models. We further propose a multiplex genome editing and preterm recloning (MAP) approach by using the rapid growth of the ground-breaking precision genome editing technology CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). With this approach, we hope to shorten the temporal requirement in generating multiple transgenic pigs, increase the survival rate of founder pigs, and generate genetically modified pigs that will more closely resemble the disease-causing mutations and recapitulate pathological features of human conditions.