Specificity of Rnai, LNA and Crispri As Loss-Of-Function Methods in Transcriptional Analysis
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SM08 Programme
SATELLITE MEETING Regulation of gene expression from a distance: exploring mechanisms The Royal Society at Chicheley Hall, home of the Kavli Royal Society International Centre Organised by Professor Wendy Bickmore and Professor Veronica van Heyningen FRS Wednesday 24 – Thursday 25 October 2012 DAY 1 DAY 2 SESSION 1 SESSION 2 SESSION 3 SESSION 4 Enhancer assays Quantitative and dynamic analysis Quantitative & dynamic analysis of Defining enhancers and their mechanisms – transgenes, genetics, and interactomes of transcription protein binding at regulatory of action Chair: Professor Nick Hastie CBE FRS Chair: elements Chair: Dr Duncan Odom Welcome by RS & lead 09.00 Professor Anne Ferguson-Smith Chair: Professor Constance Bonifer organiser The evolution of global Dynamic use of enhancers in Design rules for bacterial Massively parallel functional 09.05 enhancers 13.30 development 09.00 enhancers 13.15 dissection of mammalian enhancers Professor Denis Duboule Professor Mike Levine Dr Roee Amit Dr Rupali Patwardhan 09.30 Discussion 14.00 Discussion 09.30 Discussion 13.45 Discussion Complex protein dynamics at HERC2 rs12913832 modulates The pluripotent 3D genome Gene expression genomics eukaryotic regulatory human pigmentation by attenuating 09.45 14.15 09.45 Professor Wouter de Laat Dr Sarah Teichmann elements chromatin-loop formation between a 14.00 Dr Gordon Hager long-range enhancer and the OCA2 promoter 10.15 Discussion 14.45 Discussion 10.15 Discussion Dr Robert-Jan Palstra 10.30 Coffee 15.00 Tea 10.30 Coffee 14.15 Tea Maps of open chromatin -
A CRISPR Activation and Interference Toolkit for Industrial Saccharomyces Cerevisiae Strain KE6‑12 Elena Cámara, Ibai Lenitz & Yvonne Nygård*
www.nature.com/scientificreports OPEN A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6‑12 Elena Cámara, Ibai Lenitz & Yvonne Nygård* Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology‑directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fuorescent protein‑encoding genes under two diferent promoters allowing expression alterations up to ~ 2.5‑fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design‑build‑test cycle for developing various industrial production strains. Te yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms for industrial applications ranging from wine and beer fermentations to the production of biofuels and high-value metabolites1,2. How- ever, some of the current production processes are compromised by low yields and productivities, thus further optimization is required3. -
Quantitative CRISPR Interference Screens in Yeast Identify Chemical-Genetic Interactions and New Rules for Guide RNA Design Justin D
Smith et al. Genome Biology (2016) 17:45 DOI 10.1186/s13059-016-0900-9 RESEARCH Open Access Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design Justin D. Smith1,2, Sundari Suresh1, Ulrich Schlecht1, Manhong Wu3, Omar Wagih4, Gary Peltz3, Ronald W. Davis1, Lars M. Steinmetz1,2,5, Leopold Parts2,5,6* and Robert P. St.Onge1* Abstract Background: Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae. Results: We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets. -
High-Resolution Mapping of Transcriptional Dynamics Across Tissue Development Reveals a Stable Mrna–Trna Interface
Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Research High-resolution mapping of transcriptional dynamics across tissue development reveals a stable mRNA–tRNA interface Bianca M. Schmitt,1,5 Konrad L.M. Rudolph,2,5 Panagiota Karagianni,3 Nuno A. Fonseca,2 Robert J. White,4 Iannis Talianidis,3 Duncan T. Odom,1 John C. Marioni,2 and Claudia Kutter1 1University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, CB2 0RE, United Kingdom; 2European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, United Kingdom; 3B.S.R.C. Alexander Flemming, 16672, Vari, Athens, Greece; 4University of York, Department of Biology, Heslington, York, YO10 5DD, United Kingdom The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we characterized the expression of mRNA and tRNA genes quantitatively at multiple time points in two developing mouse tissues. We dis- covered that mRNA codon pools are highly stable over development and simply reflect the genomic background; in contrast, precise regulation of tRNA gene families is required to create the corresponding tRNA transcriptomes. The dynamic regulation of tRNA genes during development is controlled in order to generate an anticodon pool that closely corresponds to messenger RNAs. Thus, across development, the pools of mRNA codons and tRNA anticodons are invariant and highly correlated, revealing a stable molecular interaction interlocking transcription and translation. [Supplemental material is available for this article.] Transcription of the mammalian genome is divided among mul- differencesinexpressionofspecifictRNAgenesmaycontribute tiple RNA polymerases (Pol), each transcribing a nonoverlapping to tumorigenesis by favoring translation of cancer-promoting set of genes. -
DKFZ Postdoctoral Fellowships 2021 Hosting Group Information For
DKFZ Postdoctoral Fellowships 2021 Hosting group information for applicants Name of DKFZ research division/group: Division of Regulatory Genomics and Cancer Evolution (B270) Contact person: Duncan Odom Group homepage: https://www.dkfz.de/en/regulatorische-genomik/index.php Please visit our website for further information on our research and recent publications. RESEARCH PROFILE AND PROJECT TOPICS: Dr Odom’s laboratory studies how genetic sequence information shapes the cell's DNA regulatory landscape and thus the trajectory of cancer genome evolution. Our long-standing interest in interspecies analysis of matched functional genomic data has revealed the extensive and rapid turn-over of tissue-specific transcription factor binding, insulator elements, polymerase occupancies, and enhancer activities during organismal evolution. My laboratory has begun exploiting single-cell RNA-sequencing and large-scale whole genome sequencing in understanding molecular and cancer genome evolution. Recent high-profile studies from the Odom lab used single-cell transcriptional analysis to conclusively demonstrate that ageing results in substantial increases in cell-to-cell transcriptional variability and exploited chemical carcinogenesis in mouse liver to reveal how the persistence of DNA lesions profoundly shapes tumour genomes. First, we are currently interviewing for experimentally focused postdoctoral fellows to spearhead multiple diverse and interdisciplinary collaborations in single-cell analysis, functional genomics, and spatial transcriptomics with Gerstung, Stegle, Hoefer, and Goncalves laboratories at DKFZ. Such an appointment would offer unusual opportunities for an experimentally-focused postdoc to learn and deploy bioinformatics tools in collaboration with one or more world-leading computational biology laboratories. Second, we are seeking a postdoctoral researcher to spearhead a collaboration with Edith Heard's group at EMBL, focused on how X-chromosome dynamics can shape disease susceptibility in older individuals. -
CRISPR-Cas Systems Restrict Horizontal Gene Transfer In
bioRxiv preprint doi: https://doi.org/10.1101/2020.09.19.304717; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 CRISPR-Cas systems restrict horizontal gene transfer in 2 Pseudomonas aeruginosa 3 Running title: CRISPR-Cas systems in Pseudomonas aeruginosa 4 5 Authors: Rachel M. Wheatley1,* and R. Craig MacLean1 6 7 Affiliations: Department of Zoology, University of Oxford, Oxford OX1 3PS, UK 8 9 Contact details: [email protected] 10 11 Competing interests 12 This project was supported by Wellcome Trust Grant 106918/Z/15/Z held by RCM. The authors declare 13 they have no conflict of interest. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.19.304717; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 28 Abstract 29 CRISPR-Cas systems provide bacteria and archaea with an adaptive immune system that targets foreign 30 DNA. However, the xenogenic nature of immunity provided by CRISPR-Cas raises the possibility that 31 these systems may constrain horizontal gene transfer. Here we test this hypothesis in the opportunistic 32 pathogen Pseudomonas aeruginosa, which has emerged an important model system for understanding 33 CRISPR-Cas function. Across the diversity of P. aeruginosa, active CRISPR-Cas systems are 34 associated with smaller genomes and a reduced GC content, suggesting that CRISPR-Cas inhibits the 35 acquisition of foreign DNA. -
CRISPR Interference (Crispri) for Gene Regulation and Succinate Production in Cyanobacterium S
Huang et al. Microb Cell Fact (2016) 15:196 DOI 10.1186/s12934-016-0595-3 Microbial Cell Factories RESEARCH Open Access CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942 Chun‑Hung Huang, Claire R. Shen, Hung Li, Li‑Yu Sung, Meng‑Ying Wu and Yu‑Chen Hu* Abstract Background: Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence- specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various pro‑ caryotic cells. Results: To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes express‑ ing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome. Co-expression of dCas9 and sgRNA conferred effective and stable suppression of EYFP produc‑ tion at efficiencies exceeding 99%, without impairing cell growth. We next integrated the dCas9 and sgRNA targeting endogenous genes essential for glycogen accumulation (glgc) and succinate conversion to fumarate (sdhA and sdhB). Transcription levels of glgc, sdhA and sdhB were effectively suppressed with efficiencies depending on the sgRNA binding site. Targeted suppression of glgc reduced the expression to 6.2%, attenuated the glycogen accumulation to 4.8% and significantly enhanced the succinate titer. Targeting sdhA or sdhB also effectively downregulated the gene expression and enhanced the succinate titer 12.5-fold to 0.58–0.63 mg/L. -
Paterson Institute for Cancer Research Scientific Report 2008 Contents
paterson institute for cancer research scientific report 2008 cover images: Main image supplied by Karim Labib and Alberto sanchez-Diaz (cell cycle Group). Budding yeast cells lacking the inn1 protein are unable to complete cytokinesis. these cells express a fusion of a green fluorescent protein to a marker of the plasma membrane, and have red fluorescent proteins attached to components of the spindle poles and actomyosin ring (sanchez-Diaz et al., nature cell Biology 2008; 10: 395). Additional images: front cover image supplied by Helen rushton, simon Woodcock and Angeliki Malliri (cell signalling Group). the image is of a mitotic spindle in fixed MDcK (Madin-Darby canine kidney) epithelial cells, which have been stained with an anti-beta tubulin antibody (green), DApi (blue) and an anti-centromere antibody (crest, red) which recognises the kinetochores of the chromosomes. the image was taken on the spinning disk confocal microscope using a 150 x lens. rear cover image supplied by Andrei ivanov and tim illidge (targeted therapy Group). Visualisation of tubulin (green) and quadripolar mitosis (DnA stained with DApi), Burkitt’s lymphoma namalwa cell after 10 Gy irradiation. issn 1740-4525 copyright 2008 © cancer research UK Paterson Institute for Cancer Research Scientific Report 2008 Contents 4 Director’s Introduction Researchers’ pages – Paterson Institute for Cancer Research 8 Crispin Miller Applied Computational Biology and Bioinformatics 10 Geoff Margison Carcinogenesis 12 Karim Labib Cell Cycle 14 Iain Hagan Cell Division 16 Nic Jones -
RNA Interference in the Age of CRISPR: Will CRISPR Interfere with Rnai?
International Journal of Molecular Sciences Review RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi? Unnikrishnan Unniyampurath 1, Rajendra Pilankatta 2 and Manoj N. Krishnan 1,* 1 Program on Emerging Infectious Diseases, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore; [email protected] 2 Department of Biochemistry and Molecular Biology, School of Biological Sciences, Central University of Kerala, Nileshwar 671328, India; [email protected] * Correspondence: [email protected]; Tel.: +65-6516-2666 Academic Editor: Michael Ladomery Received: 2 November 2015; Accepted: 15 February 2016; Published: 26 February 2016 Abstract: The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi) based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. -
Development of CRISPR Interference (Crispri) Platform for Metabolic Engineering of Leuconostoc Citreum and Its Application for Engineering Riboflavin Biosynthesis
International Journal of Molecular Sciences Article Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis Jaewoo Son 1, Seung Hoon Jang 1, Ji Won Cha 1 and Ki Jun Jeong 1,2,* 1 Department of Chemical and Biomolecular Engineering, BK21 Plus program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea; [email protected] (J.S.); [email protected] (S.H.J.); [email protected] (J.W.C.) 2 Institute for The BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea * Correspondence: [email protected]; Tel.: +82-42-350-3934 Received: 1 July 2020; Accepted: 3 August 2020; Published: 5 August 2020 Abstract: Leuconostoc citreum, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine–Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. -
Challenges, New Technologies and Their Use in Plants
International Journal of Molecular Sciences Review Chromatin Manipulation and Editing: Challenges, New Technologies and Their Use in Plants Kateryna Fal 1,† , Denisa Tomkova 2,†, Gilles Vachon 1 , Marie-Edith Chabouté 2, Alexandre Berr 2,* and Cristel C. Carles 1,* 1 Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble Alpes, CNRS, CEA, INRAE, IRIG-LPCV, 38000 Grenoble, France; [email protected] (K.F.); [email protected] (G.V.) 2 Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg CEDEX, France; [email protected] (D.T.); [email protected] (M.-E.C.) * Correspondence: [email protected] (A.B.); [email protected] (C.C.C.) † These authors contributed equally to this work. Abstract: An ongoing challenge in functional epigenomics is to develop tools for precise manipulation of epigenetic marks. These tools would allow moving from correlation-based to causal-based findings, a necessary step to reach conclusions on mechanistic principles. In this review, we describe and discuss the advantages and limits of tools and technologies developed to impact epigenetic marks, and which could be employed to study their direct effect on nuclear and chromatin structure, on transcription, and their further genuine role in plant cell fate and development. On one hand, epigenome-wide approaches include drug inhibitors for chromatin modifiers or readers, nanobodies against histone marks or lines expressing modified histones or mutant chromatin effectors. On the other hand, locus-specific approaches consist in targeting precise regions on the chromatin, with engineered proteins able to modify epigenetic marks. -
Register of Declared Private, Professional Commercial and Other Interests
REGISTER OF DECLARED PRIVATE, PROFESSIONAL COMMERCIAL AND OTHER INTERESTS Please refer to the attached guidance notes before completing this register entry. In addition to guidance on each section, examples of information required are also provided. Where you have no relevant interests in the relevant category, please enter ‘none’ in the register entry. Name: Michaela Frye Please list all MRC bodies you are a member of: E.g. Council, Strategy Board, Research Board, Expert Panel etc and your position (e.g. chair, member). Regenerative Medicine Research Committee member Main form of employment: Name of University and Department or other employing body (include location), and your position. University of Cambridge, Department of Genetics, Cancer Research UK Senior Fellow Research group/department web page: Provide a link to any relevant web pages for your research group or individual page on your organisation’s web site. http://www.stemcells.cam.ac.uk/research/affiliates/dr-frye http://www.gen.cam.ac.uk/research-groups/frye Please give details of any potential conflicts of interests arising out of the following: 1. Personal Remuneration: Including employment, pensions, consultancies, directorships, honoraria. See section 1 for further guidance. None 2. Shareholdings and Financial Interests in companies: Include the names of companies involved in medical/biomedical research, pharmaceuticals, biotechnology, healthcare provision and related fields where shareholdings or other financial interests. See section 2 for thresholds and further guidance. None 3. Research Income during current session (financial year) : Declare all research income from bodies supported by the MRC and research income from other sources above the limit of £50k per grant for the year.