Publications by authors named "Lars Bolund"

167 Publications

The Dynamic Changes of Transcription Factors During the Development Processes of Human Biparental and Uniparental Embryos.

Front Cell Dev Biol 2021 17;9:709498. Epub 2021 Sep 17.

College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.

Previous studies have revealed that transcription factors (TFs) play important roles in biparental (BI) early human embryogenesis. However, the contribution of TFs during early uniparental embryo development is still largely unknown. Here we systematically studied the expression profiles of transcription factors in early embryonic development and revealed the dynamic changes of TFs in human biparental and uniparental embryogenesis by single-cell RNA sequencing (scRNA-seq). In general, the TF expression model of uniparental embryos showed a high degree of conformity with biparental embryos. The detailed network analysis of three different types of embryos identified that 10 out of 17 hub TFs were shared or specifically owned, such as ZNF480, ZNF581, PHB, and POU5F1, were four shared TFs, ZFN534, GTF3A, ZNF771, TEAD4, and LIN28A, were androgenic (AG) specific TFs, and ZFP42 was the only one parthenogenetic (PG) specific TF. All the four shared TFs were validated using human embryonic stem cell (hESC) differentiation experiments; most of their target genes are responsible for stem cell maintenance and differentiation. We also found that Zf-C2H2, HMG, and MYB were three dominant transcription factor families that appeared in early embryogenesis. Altogether, our work provides a comprehensive regulatory framework and better understanding of TF function in human biparental and uniparental embryogenesis.
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http://dx.doi.org/10.3389/fcell.2021.709498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8484909PMC
September 2021

Toward the massive genome of Proteus anguinus-illuminating longevity, regeneration, convergent evolution, and metabolic disorders.

Ann N Y Acad Sci 2022 Jan 3;1507(1):5-11. Epub 2021 Sep 3.

Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Qingdao, China.

Deciphering the genetic code of organisms with unusual phenotypes can help answer fundamental biological questions and provide insight into mechanisms relevant to human biomedical research. The cave salamander Proteus anguinus (Urodela: Proteidae), also known as the olm, is an example of a species with unique morphological and physiological adaptations to its subterranean environment, including regenerative abilities, resistance to prolonged starvation, and a life span of more than 100 years. However, the structure and sequence of the olm genome is still largely unknown owing to its enormous size, estimated at nearly 50 gigabases. An international Proteus Genome Research Consortium has been formed to decipher the olm genome. This perspective provides the scientific and biomedical rationale for exploring the olm genome and outlines potential outcomes, challenges, and methodological approaches required to analyze and annotate the genome of this unique amphibian.
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http://dx.doi.org/10.1111/nyas.14686DOI Listing
January 2022

Comparative analysis of clonal evolution among patients with right- and left-sided colon and rectal cancer.

iScience 2021 Jul 11;24(7):102718. Epub 2021 Jun 11.

BGI-Qingdao, BGI-Shenzhen, Qingdao 266555, China.

Tumor multiregion sequencing reveals intratumor heterogeneity (ITH) and clonal evolution playing a key role in tumor progression and metastases. Large-scale high-depth multiregional sequencing of colorectal cancer, comparative analysis among patients with right-sided colon cancer (RCC), left-sided colon cancer (LCC), and rectal cancer (RC), as well as the study of lymph node metastasis (LN) with extranodal tumor deposits (ENTDs) from evolutionary perspective remain weakly explored. Here, we recruited 68 patients with RCC (18), LCC (20), and RC (30). We performed high-depth whole-exome sequencing of 206 tumor regions including 176 primary tumors, 19 LN, and 11 ENTD samples. Our results showed ITH with a Darwinian pattern of evolution and the evolution pattern of LCC and RC was more complex and divergent than RCC. Genetic and evolutionary evidences found that both LN and ENTD originated from different clones. Moreover, ENTD was a distinct entity from LN and evolved later.
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http://dx.doi.org/10.1016/j.isci.2021.102718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254024PMC
July 2021

A porcine brain-wide RNA editing landscape.

Commun Biol 2021 06 10;4(1):717. Epub 2021 Jun 10.

Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Shenzhen, China.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is an essential post-transcriptional modification. Although hundreds of thousands of RNA editing sites have been reported in mammals, brain-wide analysis of the RNA editing in the mammalian brain remains rare. Here, a genome-wide RNA-editing investigation is performed in 119 samples, representing 30 anatomically defined subregions in the pig brain. We identify a total of 682,037 A-to-I RNA editing sites of which 97% are not identified before. Within the pig brain, cerebellum and olfactory bulb are regions with most edited transcripts. The editing level of sites residing in protein-coding regions are similar across brain regions, whereas region-distinct editing is observed in repetitive sequences. Highly edited conserved recoding events in pig and human brain are found in neurotransmitter receptors, demonstrating the evolutionary importance of RNA editing in neurotransmission functions. Although potential data biases caused by age, sex or health status are not considered, this study provides a rich resource to better understand the evolutionary importance of post-transcriptional RNA editing.
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http://dx.doi.org/10.1038/s42003-021-02238-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192503PMC
June 2021

Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning.

Nat Commun 2021 05 28;12(1):3238. Epub 2021 May 28.

Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, Qingdao, China.

The design of CRISPR gRNAs requires accurate on-target efficiency predictions, which demand high-quality gRNA activity data and efficient modeling. To advance, we here report on the generation of on-target gRNA activity data for 10,592 SpCas9 gRNAs. Integrating these with complementary published data, we train a deep learning model, CRISPRon, on 23,902 gRNAs. Compared to existing tools, CRISPRon exhibits significantly higher prediction performances on four test datasets not overlapping with training data used for the development of these tools. Furthermore, we present an interactive gRNA design webserver based on the CRISPRon standalone software, both available via https://rth.dk/resources/crispr/ . CRISPRon advances CRISPR applications by providing more accurate gRNA efficiency predictions than the existing tools.
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http://dx.doi.org/10.1038/s41467-021-23576-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163799PMC
May 2021

Efficient correction of Duchenne muscular dystrophy mutations by SpCas9 and dual gRNAs.

Mol Ther Nucleic Acids 2021 Jun 13;24:403-415. Epub 2021 Mar 13.

Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Qingdao 266555, China.

CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human transcription factor. Restoration of expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.
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http://dx.doi.org/10.1016/j.omtn.2021.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039775PMC
June 2021

Single-Cell RNA Sequencing Maps Endothelial Metabolic Plasticity in Pathological Angiogenesis.

Cell Metab 2020 04;31(4):862-877.e14

Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, VIB, Department of Oncology, Leuven Cancer Institute, KU Leuven, Leuven 3000, Belgium.

Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cell-cycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets.
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http://dx.doi.org/10.1016/j.cmet.2020.03.009DOI Listing
April 2020

An atlas of the protein-coding genes in the human, pig, and mouse brain.

Science 2020 03;367(6482)

Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden.

The brain, with its diverse physiology and intricate cellular organization, is the most complex organ of the mammalian body. To expand our basic understanding of the neurobiology of the brain and its diseases, we performed a comprehensive molecular dissection of 10 major brain regions and multiple subregions using a variety of transcriptomics methods and antibody-based mapping. This analysis was carried out in the human, pig, and mouse brain to allow the identification of regional expression profiles, as well as to study similarities and differences in expression levels between the three species. The resulting data have been made available in an open-access Brain Atlas resource, part of the Human Protein Atlas, to allow exploration and comparison of the expression of individual protein-coding genes in various parts of the mammalian brain.
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http://dx.doi.org/10.1126/science.aay5947DOI Listing
March 2020

Single-Cell Transcriptome Atlas of Murine Endothelial Cells.

Cell 2020 02 13;180(4):764-779.e20. Epub 2020 Feb 13.

Laboratory for Functional Epigenetics, Department of Human Genetics, KU Leuven, 3000 Leuven, Belgium.

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.
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http://dx.doi.org/10.1016/j.cell.2020.01.015DOI Listing
February 2020

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

Nucleic Acids Res 2020 03;48(5):e25

Department of Biomedicine, Aarhus University, Aarhus 8000, Denmark.

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.
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http://dx.doi.org/10.1093/nar/gkz1233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049710PMC
March 2020

An Integrated Gene Expression Landscape Profiling Approach to Identify Lung Tumor Endothelial Cell Heterogeneity and Angiogenic Candidates.

Cancer Cell 2020 01;37(1):21-36.e13

Laboratory for Precision Cancer Medicine, Translational Cell & Tissue Research, Department of Imaging & Pathology, KU Leuven, Leuven 3000, Belgium.

Heterogeneity of lung tumor endothelial cell (TEC) phenotypes across patients, species (human/mouse), and models (in vivo/in vitro) remains poorly inventoried at the single-cell level. We single-cell RNA (scRNA)-sequenced 56,771 endothelial cells from human/mouse (peri)-tumoral lung and cultured human lung TECs, and detected 17 known and 16 previously unrecognized phenotypes, including TECs putatively regulating immune surveillance. We resolved the canonical tip TECs into a known migratory tip and a putative basement-membrane remodeling breach phenotype. Tip TEC signatures correlated with patient survival, and tip/breach TECs were most sensitive to vascular endothelial growth factor blockade. Only tip TECs were congruent across species/models and shared conserved markers. Integrated analysis of the scRNA-sequenced data with orthogonal multi-omics and meta-analysis data across different human tumors, validated by functional analysis, identified collagen modification as a candidate angiogenic pathway.
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http://dx.doi.org/10.1016/j.ccell.2019.12.001DOI Listing
January 2020

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

J Am Soc Nephrol 2020 01 9;31(1):118-138. Epub 2019 Dec 9.

Department of Oncology, Laboratory of Angiogenesis and Vascular Metabolism, Katholieke Universiteit Leuven (KU Leuven), Leuven, Belgium;

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 and in dehydrated mice .

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.
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http://dx.doi.org/10.1681/ASN.2019080832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935008PMC
January 2020

Single-Cell Transcriptome Analysis of Uniparental Embryos Reveals Parent-of-Origin Effects on Human Preimplantation Development.

Cell Stem Cell 2019 Nov 3;25(5):697-712.e6. Epub 2019 Oct 3.

Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha 410078, China; Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha 410078, China; Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha 410078, China; National Engineering and Research Center of Human Stem Cells, Changsha 410078, China. Electronic address:

To investigate the contribution of parental genomes to early embryogenesis, we profiled the single-cell transcriptomes of human biparental and uniparental embryos systematically from the 1-cell to the morula stage. We observed that uniparental embryos exhibited variable and decreased embryonic genome activation (EGA). Comparative transcriptome analysis identified 807 maternally biased expressed genes (MBGs) and 581 paternally biased expressed genes (PBGs) in the preimplantation stages. MBGs became apparent at the 4-cell stage and contributed to the initiation of EGA, whereas PBGs preferentially appeared at the 8-cell stage and might affect embryo compaction and trophectoderm specification. Regulatory network analysis revealed that DUX4, EGR2, and DUXA are key transcription factors in MBGs' expression; ZNF263 and KLF3 are important for PBGs' expression. We demonstrated that parent-specific DNA methylation might account for the expression of most PBGs. Our results provide a valuable resource to understand parental genome activation and might help to elucidate parent-of-origin effects in early human development.
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http://dx.doi.org/10.1016/j.stem.2019.09.004DOI Listing
November 2019

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

Cell Mol Life Sci 2019 Jul 18;76(13):2633-2645. Epub 2019 Mar 18.

Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.

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.
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http://dx.doi.org/10.1007/s00018-019-03064-xDOI Listing
July 2019

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

Nucleic Acids Res 2018 12;46(22):e131

Department of Biomedicine, Aarhus University, Denmark.

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.
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http://dx.doi.org/10.1093/nar/gky767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294522PMC
December 2018

Sex Differences in Genetic Associations With Longevity.

JAMA Netw Open 2018 08 24;1(4):e181670. Epub 2018 Aug 24.

Center for the Study of Aging and Human Development, Medical School of Duke University, Durham, North Carolina (Zeng, H. Chen); Center for Healthy Aging and Development Studies, National School of Development, Raissun Institute for Advanced Studies, Peking University, Beijing, China (Zeng, Bai); BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China (Nie);BGI-Shenzhen, Shenzhen, China (Nie, Xiaomin Liu, Ye, Z. Chen, Bolund, Hou, Xiao Liu, Xu, H. Yang); The First Affiliated Hospital, Institute of Translational Medicine, School of Medicine, Zhejiang University, Hangzhou, China (Min, E. Xie); Business School of Xiangtan University, Xiangtan, China (H. Chen); Division of Non-Communicable Disease Control and Community Health, Chinese Center for Disease Control and Prevention, Beijing, China (Yin); National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing, China (Lv, Shi); Department of Sociology, Peking University, Beijing, China (Lu, Li); School of Life Sciences, Fudan University, Shanghai, China (Ni); Department of Biomedicine, Aarhus University, Aarhus, Denmark (Bolund); Duke Population Research Institute, Duke University, Durham, North Carolina (Land, Yashin, O'Rand); The MOH Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing, China (Sun, Z. Yang); School of Life Sciences, Peking University, Beijing, China (Tao, Gu); Boston University, Boston, Massachusetts (Gurinovich, Sebastiani, Perls); University of Bologna, Bologna, Italy (Franceschi); Department of Biostatistics and Bioinformatics, Duke University, Durham, North Carolina (J. Xie); French National Institute on Health and Medical Research and Ecole Pratique des Hautes Etudes, University of Montpellier, Montpellier, France (Robine); Max Planck Institute for Biology of Ageing, Cologne, Germany (Deelen); Department of Molecular Epidemiology, Leiden University Medical Center, Leiden, the Netherlands (Slagboom); Molecular Physiology Institute, Medical Center, Duke University, Durham, North Carolina (Hauser); Department of Neurology, Medical Center, Duke University, Durham, North Carolina (Gottschalk, Lutz); University of Southern Denmark, Odense, Denmark (Tan, Christensen); Human Aging Research Institute and School of Life Science, Nanchang University, Jiangxi, China (Tian); James D. Watson Institute of Genome Sciences, Hangzhou, China (H. Yang); Max Planck Institute for Demographic Research, Rostock, Germany (Vaupel).

Importance: Sex differences in genetic associations with human longevity remain largely unknown; investigations on this topic are important for individualized health care.

Objective: To explore sex differences in genetic associations with longevity.

Design Setting And Participants: This population-based case-control study used sex-specific genome-wide association study and polygenic risk score (PRS) analyses to examine sex differences in genetic associations with longevity. Five hundred sixty-four male and 1614 female participants older than 100 years were compared with a control group of 773 male and 1526 female individuals aged 40 to 64 years. All were Chinese Longitudinal Healthy Longevity Study participants with Han ethnicity who were recruited in 1998 and 2008 to 2014.

Main Outcomes And Measures: Sex-specific loci and pathways associated with longevity and PRS measures of joint effects of sex-specific loci.

Results: Eleven male-specific and 11 female-specific longevity loci ( < 10) and 35 male-specific and 25 female-specific longevity loci (10 ≤ < 10) were identified. Each of these loci's associations with longevity were replicated in north and south regions of China in one sex but were not significant in the other sex ( = .13-.97), and loci-sex interaction effects were significant ( < .05). The associations of loci rs60210535 of the gene with longevity were replicated in Chinese women ( = 9.0 × 10) and US women ( = 4.6 × 10) but not significant in Chinese and US men. The associations of the loci rs2622624 of the gene were replicated in Chinese women ( = 6.8 × 10) and European women ( = .003) but not significant in both Chinese and European men. Eleven male-specific pathways (inflammation and immunity genes) and 34 female-specific pathways (tryptophan metabolism and PGC-1α induced) were significantly associated with longevity ( < .005; false discovery rate < 0.05). The PRS analyses demonstrated that sex-specific associations with longevity of the 4 exclusive groups of 11 male-specific and 11 female-specific loci ( < 10) and 35 male-specific and 25 female-specific loci (10 ≤ < 10) were jointly replicated across north and south discovery and target samples. Analyses using the combined data set of north and south showed that these 4 groups of sex-specific loci were jointly and significantly associated with longevity in one sex ( = 2.9 × 10 to 1.3 × 10) but not jointly significant in the other sex ( = .11 to .70), while interaction effects between PRS and sex were significant ( = 4.8 × 10 to 1.2 × 10).

Conclusion And Relevance: The sex differences in genetic associations with longevity are remarkable, but have been overlooked by previously published genome-wide association studies on longevity. This study contributes to filling this research gap and provides a scientific basis for further investigating effects of sex-specific genetic variants and their interactions with environment on healthy aging, which may substantially contribute to more effective and targeted individualized health care for male and female elderly individuals.
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http://dx.doi.org/10.1001/jamanetworkopen.2018.1670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173523PMC
August 2018

Apolipoprotein E Deficiency Increases Remnant Lipoproteins and Accelerates Progressive Atherosclerosis, But Not Xanthoma Formation, in Gene-Modified Minipigs.

JACC Basic Transl Sci 2017 Oct 30;2(5):591-600. Epub 2017 Oct 30.

Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.

Deficiency of apolipoprotein E (APOE) causes familial dysbetalipoproteinemia in humans resulting in a higher risk of atherosclerotic disease. In mice, APOE deficiency results in a severe atherosclerosis phenotype, but it is unknown to what extent this is unique to mice. In this study, was targeted in Yucatan minipigs. minipigs displayed increased plasma cholesterol and accumulation of apolipoprotein B-48-containing chylomicron remnants on low-fat diet, which was significantly accentuated upon feeding a high-fat, high-cholesterol diet. minipigs displayed accelerated progressive atherosclerosis but not xanthoma formation. This indicates that remnant lipoproteinemia does not induce early lesions but is atherogenic in pre-existing atherosclerosis.
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http://dx.doi.org/10.1016/j.jacbts.2017.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058916PMC
October 2017

The correlation of copy number variations with longevity in a genome-wide association study of Han Chinese.

Aging (Albany NY) 2018 Jun;10(6):1206-1222

BGI Shenzhen, Shenzhen 518083, China.

Copy number variations (CNVs) have been shown to cause numerous diseases, however, their roles in human lifespan remain elusive. In this study, we investigate the association of CNVs with longevity by comparing the Han Chinese genomes of long-lived individuals from 90 to 117 years of age and the middle-aged from 30 to 65. Our data demonstrate that the numbers of CNVs, especially deletions, increase significantly in a direct correlation with longevity. We identify eleven CNVs that strongly associate with longevity; four of them locate in the chromosome bands, 7p11.2, 20q13.33, 19p12 and 8p23.3 and overlap partially with the CNVs identified in long-lived Danish or U.S. populations, while the other seven have not been reported previously. These CNV regions encode nineteen known genes, and some of which have been shown to affect aging-related phenotypes such as the shortening of telomere length (), the risk of cancer (), and vascular and immune-related diseases (). In addition, we found several pathways enriched in long-lived genomes, including FOXA1 and FOXA transcription factor networks involved in regulating aging or age-dependent diseases such as cancer. Thus, our study has identified longevity-associated CNV regions and their affected genes and pathways. Our results suggest that the human genome structures such as CNVs might play an important role in determining a long life in human.
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http://dx.doi.org/10.18632/aging.101461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6046244PMC
June 2018

WEGO 2.0: a web tool for analyzing and plotting GO annotations, 2018 update.

Nucleic Acids Res 2018 07;46(W1):W71-W75

Department of Oncology, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China.

WEGO (Web Gene Ontology Annotation Plot), created in 2006, is a simple but useful tool for visualizing, comparing and plotting GO (Gene Ontology) annotation results. Owing largely to the rapid development of high-throughput sequencing and the increasing acceptance of GO, WEGO has benefitted from outstanding performance regarding the number of users and citations in recent years, which motivated us to update to version 2.0. WEGO uses the GO annotation results as input. Based on GO's standardized DAG (Directed Acyclic Graph) structured vocabulary system, the number of genes corresponding to each GO ID is calculated and shown in a graphical format. WEGO 2.0 updates have targeted four aspects, aiming to provide a more efficient and up-to-date approach for comparative genomic analyses. First, the number of input files, previously limited to three, is now unlimited, allowing WEGO to analyze multiple datasets. Also added in this version are the reference datasets of nine model species that can be adopted as baselines in genomic comparative analyses. Furthermore, in the analyzing processes each Chi-square test is carried out for multiple datasets instead of every two samples. At last, WEGO 2.0 provides an additional output graph along with the traditional WEGO histogram, displaying the sorted P-values of GO terms and indicating their significant differences. At the same time, WEGO 2.0 features an entirely new user interface. WEGO is available for free at http://wego.genomics.org.cn.
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http://dx.doi.org/10.1093/nar/gky400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030983PMC
July 2018

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases.

Gigascience 2018 03;7(3):1-19

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

Background: Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated.

Findings: We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription.

Conclusion: Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.
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http://dx.doi.org/10.1093/gigascience/giy011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888497PMC
March 2018

Central and Peripheral Nervous System Progenitors Derived from Human Pluripotent Stem Cells Reveal a Unique Temporal and Cell-Type Specific Expression of PMCAs.

Front Cell Dev Biol 2018 6;6. Epub 2018 Feb 6.

Danish Research Institute of Translational Neuroscience, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark.

The P-type ATPases family consists of ion and lipid transporters. Their unique diversity in function and expression is critical for normal development. In this study we investigated human pluripotent stem cells (hPSC) and different neural progenitor states to characterize the expression of the plasma membrane calcium ATPases (PMCAs) during human neural development and in mature mesencephalic dopaminergic (mesDA) neurons. Our RNA sequencing data identified a dynamic change in ATPase expression correlating with the differentiation time of the neural progenitors, which was independent of the neuronal progenitor type. Expression of ATP2B1 and ATP2B4 were the most abundantly expressed, in accordance with their main role in Ca regulation and we observed all of the PMCAs to have a subcellular punctate localization. Interestingly in hPSCs ATP2B1 and ATP2B3 were highly expressed in a cell cycle specific manner and ATP2B2 and ATP2B4 were highly expressed in a hPSC sub-population. In neural rosettes a strong apical PMCA expression was identified in the luminal region. Lastly, we confirmed all PMCAs to be expressed in mesDA neurons, however at varying levels. Our results reveal that PMCA expression dynamically changes during stem cell differentiation and highlights the diverging needs of cell populations to regulate and properly integrate Ca changes, which can ultimately correspond to changes in specific stem cell transcription states.
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http://dx.doi.org/10.3389/fcell.2018.00005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808168PMC
February 2018

BS-virus-finder: virus integration calling using bisulfite sequencing data.

Gigascience 2018 01;7(1):1-7

Bioinformatics Research Center, Aarhus University, C. F. Møllers Allé 8, DK-8000, Aarhus C, Denmark.

Background: DNA methylation plays a key role in the regulation of gene expression and carcinogenesis. Bisulfite sequencing studies mainly focus on calling single nucleotide polymorphism, different methylation region, and find allele-specific DNA methylation. Until now, only a few software tools have focused on virus integration using bisulfite sequencing data.

Findings: We have developed a new and easy-to-use software tool, named BS-virus-finder (BSVF, RRID:SCR_015727), to detect viral integration breakpoints in whole human genomes. The tool is hosted at https://github.com/BGI-SZ/BSVF.

Conclusions: BS-virus-finder demonstrates high sensitivity and specificity. It is useful in epigenetic studies and to reveal the relationship between viral integration and DNA methylation. BS-virus-finder is the first software tool to detect virus integration loci by using bisulfite sequencing data.
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http://dx.doi.org/10.1093/gigascience/gix123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788064PMC
January 2018

Isolation and whole genome sequencing of fetal cells from maternal blood towards the ultimate non-invasive prenatal testing.

Prenat Diagn 2017 12;37(13):1311-1321

BGI-Shenzhen, Shenzhen, 518083, PR China.

Objective: The purpose of this study were to develop a methodology of isolating fetal cells from maternal blood and use deep sequence demonstrating the promise for complete and accurate genetic screening compared to other non-invasive prenatal testing.

Methods: Here in this study, we developed a double negative selection (DNS) procedure to unbiasedly enrich fetal cells. After validated by short tandem repeat (STR), the isolated circulating fetal cells (CFCs) were subjected to deep whole genome sequencing analysis.

Results: Our DNS protocol significantly increasing the purity of the mimic fetal cells from 1 in 1 million nucleated cells in whole blood to 1:8 to 1:30 (12.5%-3.33%) after 2 steps of enrichment. Isolated single fetal cell obtained a coverage rate (86.8%) and allelic dropout rate (24.90%) comparative to the reported results of human cell line. Several disease-associated variants were identified in the whole genome sequencing data of isolated CFCs and further confirmed in the sequencing data of unamplified gDNA.

Conclusion: In conclusion, the robustness of DNS and STR to collect CFCs from peripheral maternal blood for the first time coupled with deep sequencing technique demonstrates the possibility of comprehensive non-invasive prenatal testing of genetic disorders using isolated CFCs.
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http://dx.doi.org/10.1002/pd.5186DOI Listing
December 2017

Sequencing and de novo assembly of 150 genomes from Denmark as a population reference.

Nature 2017 08 26;548(7665):87-91. Epub 2017 Jul 26.

Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm 17177, Sweden.

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.
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http://dx.doi.org/10.1038/nature23264DOI Listing
August 2017

Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1.

Dis Model Mech 2017 07;10(7):869-880

Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology.
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http://dx.doi.org/10.1242/dmm.028662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536904PMC
July 2017

Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency.

FEBS Lett 2017 07 28;591(13):1892-1901. Epub 2017 Jun 28.

Department of Biomedicine, Aarhus University, Denmark.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) systems have emerged as the method of choice for genome editing, but large variations in on-target efficiencies continue to limit their applicability. Here, we investigate the effect of chromatin accessibility on Cas9-mediated gene editing efficiency for 20 gRNAs targeting 10 genomic loci in HEK293T cells using both SpCas9 and the eSpCas9(1.1) variant. Our study indicates that gene editing is more efficient in euchromatin than in heterochromatin, and we validate this finding in HeLa cells and in human fibroblasts. Furthermore, we investigate the gRNA sequence determinants of CRISPR-Cas9 activity using a surrogate reporter system and find that the efficiency of Cas9-mediated gene editing is dependent on guide sequence secondary structure formation. This knowledge can aid in the further improvement of tools for gRNA design.
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http://dx.doi.org/10.1002/1873-3468.12707DOI Listing
July 2017

RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts.

Cell Reprogram 2017 06;19(3):189-198

2 Danish Regenerative Engineering Alliance for Medicine, Department of Biomedicine, Aarhus University , Aarhus, Denmark .

Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts). Activation potential was monitored using a luciferase reporter system and we found that both CRISPRa and SAM can efficiently activate the proximal promoter of all five genes. We also observed that the guide RNA (gRNA) target sites and number of gRNAs have a major effect on gRNA-guided activation efficiency. Furthermore, increased activation efficiency (>3-folds) could be achieved by the SAM system compared to CRISPRa. In addition, we discovered that only the SAM system could efficiently activate LIN28, OCT4, and SOX2 expression (up to 100-folds compared to coexpression with a scrambled gRNA) in primary human fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular reprogramming using ATFs.
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http://dx.doi.org/10.1089/cell.2017.0006DOI Listing
June 2017

Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells.

J Biotechnol 2017 Apr 1;247:42-49. Epub 2017 Mar 1.

Department of Biomedicine, Aarhus University, Aarhus, Denmark. Electronic address:

Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells.
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http://dx.doi.org/10.1016/j.jbiotec.2017.02.024DOI Listing
April 2017
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