Today, we have joined forces to map the complex brain, which is what the brain is, in a new comparative atlas. The study has just been published in the journal Science and expands our understanding of the cerebellum as the base for coordinating motor functions. See the full news here: 

health.au.dk/en/

The breakthrough in single cell sequencing technology now allows us to study somatic cell evolution and heterogeneity in speed and scale. This technology is based on the high-throughput generation of barcoded transcripts of individual cell. Combining with next generation sequencing technology, it allows us to profile the expression of all genes cell by cell. Based on the overall expression similarity and cell identity genes (which is also known as marker genes), this technology empowers the identification of classical and novel cell types, as well as novel phenotypes (cells, genes, pathways, metabolites) which response to e.g. treatments, allowing the development of more targeted treatment of human diseases. 

One particular type of cells which plays indispensable role in human health and disease are the endothelial cells, which line the lumen of vascular systems. The phenotypic heterogeneity and metabolic reprogramming of endothelial cells across different vessel beds have been well-recognized, but the phenotypic heterogeneity of the endothelial cells at single cell level has not been inventoried in sufficient power. In collaboration with Prof. Peter Carmeliet from VIB, we have applied single cell RNA sequencing to reveal the heterogeneity and metabolic adaptation of endothelial cells in lung cancers and kidney compartments.

Read the full articles here:

1. Goveia J, Rohlenova K, Taverna F, et al. An Integrated Gene Expression Landscape Profiling Approach to Identify Lung Tumor Endothelial Cell Heterogeneity and Angiogenic Candidates. Cancer Cell. 2020;37(1):21–36.e13. doi:10.1016/j.ccell.2019.12.001 

2. Dumas SJ, Meta E, Borri M, et al. Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation. J Am Soc Nephrol. 2020;31(1):118–138. doi:10.1681/ASN.2019080832 

3. Highlights of the Renal EC study by Nature Reviews Nephrology: www.nature.com/articles/s41581-020-0250-4

Assistant Professor and PhD Lin Lin from the Department of Biomedicine receives almost DKK six million from the Independent Research Fund Denmark. The grant will be used to develop new technologies for pig-to-human organ transplants. The support from the Independent Research Fund Denmark’s Sapere Aude programme also gives her the opportunity to establish herself as research group leader. 

2018.12.07 | SABINA BJERRE HANSEN

【Aug. 29. 2018】

Dysregulated intracellular pH is emerging as a hallmark of cancer. In spite of their acidic environment and increased acid production, cancer cells maintain alkaline intracellular pH that promotes cancer progression by inhibiting apoptosis and increasing glycolysis, cell growth, migration, and invasion. Here we identify signal transducer and activator of transcription-3 (STAT3) as a key factor in the preservation of alkaline cytosol. STAT3 associates with the vacuolar H⁺-ATPase in a coiled-coil domain-dependent manner and increases its activity in living cells and in vitro. Accordingly, STAT3 depletion disrupts intracellular proton equilibrium by decreasing cytosolic pH and increasing lysosomal pH, respectively. This dysregulation can be reverted by reconstitution with wild-type STAT3 or STAT3 mutants unable to activate target genes (Tyr705Phe and DNA-binding mutant) or to regulate mitochondrial respiration (Ser727Ala). Upon cytosolic acidification, STAT3 is transcriptionally inactivated and further recruited to lysosomal membranes to reestablish intracellular proton equilibrium. These data reveal STAT3 as a regulator of intracellular pH and, vice versa, intracellular pH as a regulator of STAT3 localization and activity.

Article is published in Cell Research. 

Liu B., Palmfeldt J., Lin L., Colaço A., Clemmensen KKB., Huang J., Xu F., Liu X., Maeda K., Luo Y., Jäättelä M. STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH.Cell Res. 2018 Aug 20. doi: 10.1038/s41422-018-0080-0. [Epub ahead of print]

https://www.nature.com/articles/s41422-018-0080-0

www1.bio.ku.dk/nyheder/pressemeddelelser/populaer-gen-saks-kan-bruges-til-at-efterligne-kraeftceller/

GENTEKNOLOGI

Et dansk forskningsstudie viser, at CRISPR-teknologien, som kan klippe og klistre i gener, kan bruges til at danne cirkelformet DNA. Denne form for DNA kan bære på kræftgener, så den nye viden kan være central i forståelsen af, hvordan kræft opstår.

I laboratorier verden over anvendes I dag en teknologi kaldet CRISPR, som gør det muligt at klippe uønskede gener ud og sætte nye ind. Nu har forskere fra Aarhus Universitet og Københavns Universitet, som de førstevist, at man kan danne cirkelformet DNA i celler ved at bruge disse ’gen-sakse’.

-  ”CRISPR et kærkomment værktøj, som nu tillader meget mere nærgående studier af DNA-cirklers indflydelse på celler og mulige akkumulering over tid i levende celler. Desuden har vi med CRISPR vist, at store ring-kromosomer kan dannes med relativ høj frekvens, hvilket baner vej for mere dybdegående undersøgeler af ring-kromosomer i menneskeceller, der indtil nu har været begrænset til eksempler fra patienter med sådanne kromosomale anormaliteter”, forklarer Yonglun Luo fra Aarhus Universitet som, sammen med Birgitte Regenberg og Henrik Devitt Møller fra Københavns Universitet, står bag undersøgelsen. Deres resultater er netop publiceret i det internationale tidsskrift, Nucleic Acids Research.

Forskerne ved, at ca. halvdelen af alle tilfælde af kræft indeholder ringformet DNA elementer. Indtil nu har kræftforskere manglet metoder til at danne cirkulært DNA og undersøge, hvordan en rask celle bliver til en kræftcelle, når en cirkel dannes. Ved at klippe i et kromosom med CRISPR, kan forskerne nu danne et cirkulært DNA i en rask celle for at lære mere om deres betydning.

- ”Disse resultater er helt centrale for at forstå, hvordan kræft opstår., siger Birgitte Regenberg fra Københavns Universitet. Og hendes kollega Henrik Devitt Møller uddyber, ”Foruden kræft kan vi også studere, hvordan cirkulært DNA ellers påvirker vores krop. Vi har tidligere på året vist, at raske mennesker også bærer cirkulært DNA i cellerne, men hvad cirkulært DNA uden kræftgener gør, ved vi meget lidt om’.

Denne forskning kan på sigt få afgørende betydning for udvikling af mere effektiv medicin mod kræft og til at forstå den cirkulære DNAs funktion i cellen. Og den kan også være med til belyse, hvordan laboratorieteknikker – ved brug af CRISPR metoden - mest hensigtsmæssigt kan benyttes.

Forskningen er støttet af Carlsbergfonden, Lundbeckfonden og Danmarks Frie Forskningsfond.

Link to Article:

https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gky767/5078801

Henrik Devitt Møller, Lin Lin, Xi Xiang, Trine Skov Petersen, Jinrong Huang, Luhan Yang, Eigil Kjeldsen, Uffe Birk Jensen, Xiuqing Zhang, Xin Liu, Xun Xu, Jian Wang, Huanming Yang, George M Church, Lars Bolund, Birgitte Regenberg,  Yonglun Luo. CRISPR-C: circularization of genes and chromosome by CRISPR in human cells. Nucleic Acids Research, gky767, doi.org/10.1093/nar/gky767. 

One of the most beautiful features of the CRISPR-Cas9 system is its flexibility. By introducing two inactivating mutations to the nuclease catalytic domains of the Cas9 protein, the activity of cleaving DNA by Cas9 is inactivated, which is commonly referred as dead Cas9, or dCas9. However, dCas9 is not completely "dead". It is can be directed to a specific genomic locus via a small guide RNA (gRNA). Furthermore, by fusing dCas9 to another protein or domains, dCas9 can be engineered to acquire a new function. One such application is fusing dCas9 to the epigenetic regulating enzymes. 

Aberrant DNA methylation have long been discovered to be the cause of many human diseases, including cancers. During the last few years, we have been working on engineering the CRISPR system for targeted DNA methylation to inactivate oncogenes in cancers. In our recently study published in GigaScience, we reported generating two generations of CRISPR dCas9 methyltransferases, named as CRISPRme. Using a 3-5 gRNAs, we shown that CRISPRme can efficiently methylation and inhibit oncogenes in cancer cell lines. 

To fully characterize the specificity of CRISPRme, we conducted whole-genome bisulfide sequencing of the CRISPRme treated cells. We discovered that there are certain genomic hot spots that are prone to be unspecifically methylated, including euchromatin, promoter, 5'UTR, and CpG islands. The discovery of these hot spots will enable us to develop a next generation CRISPRme which will be more specific and efficient, as well as presenting a potential role for cancer therapy and for studying of functional epigenetics. 

Re. Article:

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

Lin Lin Yong Liu Fengping Xu Jinrong Huang Tina Fuglsang Daugaard Trine Skov Petersen Bettina Hansen Lingfei Ye Qing ZhouFang Fang Ling Yang Shengting Li Lasse Fløe Kristopher Torp Jensen Ellen Shrock Fang Chen Huanming Yang Jian Wang Xin LiuXun Xu  Lars Bolund Anders Lade Nielsen Yonglun Luo Author Notes

GigaScience, Volume 7, Issue 3, 1 March 2018, giy011, https://doi.org/10.1093/gigascience/giy011

The DREAM team have created a new SpCas9 variant with enhanced gene editing efficiency. This Cas9 variant was created by postdoc fellow Lin Lin from the DREAM team. To enhance gene editing efficiency, the SpCas9 protein was fused to E.Coli Recombinant protein A (RecA), an enzyme involved in DNA damage repair pathway in materials. Our group has shown that, fusing RecA to the SpCas9 can fine-tune the double-strand DNA damage repair pathway in mammalian cells towards non-homologous end joining. This system is useful for gene editing applications such gene knockout or deletion. The study has been accepted by the Journal of Biotechnology. This work was associated to the supports from the Lundbeck Foundation, the Danish Research Council and the Innovation Fund Denmark (BrainStem). http://www.sciencedirect.com/science/article/pii/S0168165617300871

The BrainStem and DREAM research groups describe a patient iPSC-derived neuronal model for FTD3. This cellular model shows endosome abnormalities previously reported in patients. Furthermore, it provides insights into the role of impaired mitochondria function and imbalanced iron homeostasis in FTD3 pathology. All observed phenotypes were rescued in CRISPR/Cas9-edited isogenic controls.

Patient iPSC-Derived Neurons for Disease Modeling of Frontotemporal Dementia with Mutation in CHMP2B

Yu Zhang, Benjamin Schmid, Nanett K. Nikolaisen, Mikkel A. Rasmussen, Blanca I. Aldana, Mikkel Agger, Kirstine Calloe, Tina C. Stummann, Hjalte M. Larsen, Troels T. Nielsen, Jinrong Huang, Fengping Xu, Xin Liu, Lars Bolund, Morten Meyer, Lasse K. Bak, Helle S. Waagepetersen, Yonglun Luo, Jørgen E. Nielsen, The FReJA Consortium, Bjørn Holst, Christian Clausen, Poul Hyttel, Kristine K. Freude,

Stem Cell Reports. http://dx.doi.org/10.1016/j.stemcr.2017.01.012

A special issue with focuses on genome editing in stem cells is now opened for paper submission at the journal "Stem Cell International". You can find more information about this issue through the following hyperlink: https://www.hindawi.com/journals/sci/si/402561/cfp/

Authors can submit their manuscripts through the Manuscript Tracking System at mts.hindawi.com/submit/journals/sci/gesc/.

Manuscript Due Friday, 23 June 2017 First Round of Reviews Friday, 15 September 2017 Publication Date Friday, 10 November 2017

Lead Guest Editor Yonglun Luo, Aarhus University, Aarhus, Denmark

Guest Editors Laurent Roybon, Lund University, Lund, Sweden Kristine Freude, Copenhagen University, Copenhagen, Denmark Guangqian Zhou, Shenzhen University, Shenzhen, China

(Jan 14, 2016) DURING the last three years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 system (CRISPR/Cas9) has rapidly become the most promising and powerful genome editing tool. With great efforts and contribution from numerous scientists around the world, the CRISPR/Cas9 system has now been developed and tested in almost all scenarios of genome and epigenome editing applications, such loss-of-function by targeted gene disruption, gain-of-function by targeted gene insertion, targeted gene repression or activation, epigenome editing etc.

Supported by the Danish Research Council for Independent Research and the Lundbeck Foundation, the DREAM team, lead by Associate Professor Yonglun Luo, is endeavoring to establish, improve and apply the CRISPR/Cas9 system for targeted gene editing and activation for studying the reprogramming and differentiation of human induced pluripotent stem cells. Furthermore, the DREAM team is also developing methods that can targeted modified the DNA methylome, which give great potential for studying stem cell reprogramming, differentiation and cancer therapy.

One challenge in the application of the CRISPR/Cas9 system is the validation and selection of functionally active small guide RNAs (sgRNA), which can efficiently direct the Cas9 or catalytically inactive Cas9 (dCas9) to the correct genomic loci. This week, the DREAM team published their dual-fluorescent system, named C-Check, in the scientific journal Cellular and Molecular Life Sciences [1]. The C-Check system contains two truncated and inactive green fluorescent genes (GFP), which contains homology sequences of 500 base pairs. Between the two truncated GFP genes, the C-Check vector contains a Golden-gate cloning site, where any target site can be inserted. Upon CRISPR/Cas9-mediated cleavage in the target site in the C-Check vector, the truncated GFP genes can be repaired by the single-strand annealing mechanism in the cells, which leads to the expression of a functional GFP protein (Illustrative Figure). With this system, the group demonstrated that sgRNAs, which are validated by the C-Check vectors, are also functional active at the endogenous genomic loci.

For more information about the C-Check vector, please read the related publication:

[1] Zhou et al. 2016. Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system. Cell Mol Life Sci. 2016 Jan 11. [Epub ahead of print]. PMID: 26755436

Associate Professor Yonglun Luo's group is now partner of the BrainStem – Stem Cell Center of Excellence in Neurology. The BrainStem is supported by the Innovation Fund Denmark, which will do research in neurodegenerative diseases over a 6 year period - from 2015 until 2020.

Associate Professor Yonglun Luo (Alun) is the invited speaker at the 13^thTransgenic Technology (TT2016) meeting in Prague, Czech Republic, from March 20^th-23^rd, 2016.