Welcome to The Conference
The conference addressed applications of genome editing in a variety of biological systems, featuring:
- A high-impact, packed day of talks, discussions and several hours of networking
- Oral presentations on latest developments in the field of genome editing by an international faculty of leading researchers from academia and industry
- Update on CRISPR patents
- Posters
- Trade exhibition
- Excellent networking opportunities, and a relaxed and friendly environment
Podium Agenda
Friday 5th April 2019 | The Jarvis Doctorow Hall | St Edmund Hall | Oxford, UK
0830: Registration, welcome coffee and networking
0925: Welcome and housekeeping
Session 1: Chair Ed Bolt
0930: Dr Andrea Pellagatti, Radcliffe Department of Medicine, University of Oxford, Oxford, UK
Modelling of myeloid malignancies using CRISPR/Cas9 gene editing and induced pluripotent stem cell technology
0950: Dr Leopold Parts, The Wellcome Trust Sanger Institute, Hinxton, UK
Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence
1010: Dr Cornelia Hampe, Takara Bio Europe, France
A fast and reliable method for detecting single base editing
1030: Dr Adam Corner, Bio-Rad, UK
Ultra-Sensitive Quantification of Genome Editing Events Using Droplet Digital PCR
1050: Refreshment break, exhibition, posters and networking
Session 2: Chair Marcus Lee
1130: Dr Ed Bolt, University of Nottingham, Nottingham, UK
Development of a sgRNA– targeted DNA integration protein that short – circuits homology dependent DNA repair
1150: Dr Chris Vakulskas, Integrated DNA Technologies, USA
Engineered AsCas12a variants with enhanced activity and broadened PAM compatibility
1210: Dr Emmanouil Metzakopian, The Wellcome Sanger Institute, Hinxton, UK
Genome-scale CRISPRa screen identifies novel factors for cellular reprogramming
1230: Dr Inês Cebola, National Institute for Health Research (NIHR) Imperial Biomedical Research Centre, London, UK
Validation of diabetes 3D enhancer-promoter interactions using optimised genome editing tools
1250: Lunch (provided), exhibition, posters and networking
Session 3: Chair Andrea Pellagatti
1350: Dr Philip Webber, Dehns Patent and Trade Mark Attorneys, Oxford, UK
CRISPR Patent Wars Update: Cas9 and Cpf1
1410: Dr Benoit Giquel, Addgene, UK
CRISPR plasmid technology and trends: Data from Addgene
1425: Dr Richard Jones, Merck, UK
CRISPR: synthetic guide strategies and new developments from Merck
1440: Dr Marcus Lee, The Wellcome Trust Sanger Institute, UK
CRISPR approaches for antimalarial target discovery
1500: Mr Kyros Kyrou, Imperial College London, London, UK
Temporal control of CRISPR can eliminate end-joining in favour of homologous repair. Gene drives for genetic control of the malaria mosquito
1530: Refreshment break, exhibition, posters and networking
Session 4: Chair Chris Vakulskas
1610: Dr Jose Gutierrez-Marcos, School of Life Sciences, University of Warwick, Coventry, UK
Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in plants using CRISPR/Cas9
1630: Dr Yongxiu Yao, The Pirbright Institute, Surrey, UK
Genome editing of avian herpesviruses for studying viral pathogenesis and developing recombinant vaccines
1650: Dr Sakari Vanharanta, MRC Cancer Unit, University of Cambridge, Cambridge, UK
Deconstructing transcriptional mechanisms of cancer progression
1710: Dr Sumana Sharma, EMBL-EBI, Wellcome Genome Campus, Cambridgeshire, UK
Dissecting context dependent cancer signalling processes using CRISPR-based approaches
1730: Dr Dimitrios Garyfallos, University of Cambridge, Cambridge, UK
Functional genomic studies of cancer immune evasion using in vitro and in vivo CRISPR/Cas9 genetic screens
1745: Discussion and close
Posters
Generation and validation of increasingly complex alleles introduced by genome editing
Alasdair J. Allan; Francesca J. Pike; Matthew Mackenzie; Gemma F. Codner and Lydia Teboul
The Mary Lyon Centre, MRC Harwell Institute, Harwell Campus, Didcot, Oxon, OX11 0RD, UK
Mouse models are valuable tools to understand gene function, genetic disease mechanisms and to develop and test new therapeutic treatments in vivo. The CRISPR/Cas9 system can be utilised as a genome-engineering tool and has brought new perspectives in the generation of mouse models of human disease. The use of this system allows for the introduction of targeted point mutations and other subtle modifications into the mouse genome in a time-efficient and cost-effective manner. We will present our recent developments of processes for genome engineering. As the CRISPR/Cas9 system becomes better understood and communicated, the requests for mouse models of human disease become ever more complex. These include flanking a target exon with FLOX sites, large megabase deletions of DNA sequence containing multiple genes and humanising sections of a gene to better mimic a disease model in humans. Alongside the generation of these mutants, their validation represents a new challenge. With new processes for allele validation, we uncover further variability in the outcome of applying CRISPR/Cas9 to the modification of mouse early embryos. We will demonstrate how we have used droplet digital PCR (ddPCR) to identify discrete sequence changes, the generation of larger than expected deletions and chromosomal rearrangements. Extensive validation in this manner recognises unwanted variants at early stages of the mutagenesis process, which reduces the number of animals required for genome engineering and contributes to experimental reproducibility of the model.
Development of CRISPR/Cas9-based gene drive approaches to prion disease resistance
Andrew R Castle1,2, Serene L Wohlgemuth1,2, and David Westaway1,2,3
1Centre for Prions and Protein Folding Diseases
2Department of Medicine, and
3Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada
Prion diseases are fatal, transmissible neurodegenerative disorders of mammals. They depend upon a postranslational conversion event that transforms the cellular glycoprotein PrPC, encoded by the Prnp gene, into a disease-associated conformation. One example is chronic wasting disease (CWD), which is highly infectious and is threatening cervid populations in the US and Canada. CWD cases have also been detected in Norway, Finland and South Korea. Given mixed evidence from animal studies, the possibility of zoonotic transmission of CWD cannot be ruled out and, unfortunately, there are no effective interventions for CWD, nor other prion diseases. Ongoing attempts to develop a CWD vaccine suffered a recent setback when a potential vaccine accelerated disease onset in a small-scale trial in elk. Here, as an alternative, we propose using CRISPR/Cas9-based gene drive technology to eliminate prion disease susceptibility from cervid populations. Specifically, the gene drive would promote the spread of null alleles of Prnp; knockout of this gene is known to confer absolute resistance to prion diseases, whilst loss of the encoded protein is generally well-tolerated. We are performing proof-of-principle experiments to validate this approach using rodent models. Firstly, we demonstrated that co-expression of Cas9 and murine Prnp gRNAs in RK13 cells creates indels detectable by a T7E1 assay. Secondly, we showed that electroporation of Cas9/gRNA ribonucleoprotein complexes into fertilised mouse oocytes results in pups with heterozygous Prnp disruptions. Thirdly, we have integrated sequences encoding a fluorescent reporter into the Cas9 cleavage site within Prnp by co-transfecting N2a cells with Cas9/gRNA expression plasmids and a donor plasmid containing the reporter sequences flanked by homology arms of ~800 bp. Work is ongoing to utilize inducible forms of Cas9 as gene brake mechanisms and to generate transgenic mice expressing Cas9 from germline-specific promoters.
Investigation of the effect of a dUTPASE knock out mouse line creating by CRISPR Cas9 technology
Zoltán Gál1,2, Hajnalka Laura Pálinkás3,4, Gergely Rácz3,5,Gergely Tihanyi3,5,Bálint Biró1,2, Tímea Pintér1,Elen Gócza1,OrsolyaIvett Hoffmann1, László Hiripi1, Beáta G. Vértessy3,4,5
1Agricultural Biotechnology Institute, Department of Animal Biotechnology, Szent-Györgyi Albert u. 4., 2100 Gödöllő, Hungary
2Faculty of Agricultural and Enviromental Science, SzentIstván University,Práter Károly u. 1.,2100, Gödöllő, Hungary
3Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósokkrt. 2., 1117 Budapest, Hungary
4Doctoral School of Multidisciplinary Medical Science, University of Szeged, Dóm tér 9., 6720 Szeged, Hungary
5Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, SzentGellérttér 4., 1111 Budapest, Hungary
Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is an enzyme catalyzing the reaction of the transformation of dUTP into dUMP, providing dUMP as the substrate for thymidylate synthase. This reaction promoting the removal of dUTP from the nucleotide pool and preclude the uracil genomic integration, thus preventing the cells from degradation. Despite its physiological significance dUTPase gene targeting experiments have only been performed in unicellular organisms, such as bacteria and yeast. Our aim was to produce a useful model to investigate the physiological role of the dUTPase in vivo in mammals. We have created a dUTPase knock out mouse line harbouring a 47 base pair deletion in the coding region of the DUT gene with CRISPR Cas9 technology. Heterozygous dut +/- animals are viable and fertile with decreased dUTPase level. Breeding heterozygous animals resulted no homozygous knock out siblings. Genotypes were verified by PCR technology. Homozygous embryos die in vivo shortly after implantation (before E8.5). Blastocyst stage embryos show decreased cell number both in the inner cell mass and trophectoderm. Our results pointed out that the dUTPase is essential for the proper early embryonic development. Additional combined knock-out experiments may potentially rescue the dut -/- phenotype which are in progress now to address and analyze the dut -/- phenotype. This work was supported by the National Research, Development and Innovation Office of Hungary (K109486, K119493, NVKP_16-1-2016-0020, 2017-1.3.1-VKE-2017-00002, 2017-1.3.1-VKE-2017-00013, VEKOP-2.3.2-16-2017-00013 to BGV); GÉNNET_21 (VEKOP-2.3.2-16-2016-00012 to EG); OTKA PD 111964 and OTKA 124708 to OIH.HLP was also supported by the UNKP-17-3 New National Excellence Program of the Ministry of Human Capacities.
Functional genomic studies of cancer immune evasion using in vitro and in vivo CRISPR/Cas9 genetic screens
Dimitrios A. Garyfallos1,2, Etienne De Braekeleer1,3, MS Vijayabaskar1, Hannes Ponstingl1, Sumana Sharma1,5, Kärt Tomberg1,2, George S. Vassiliou1,2,3,4 and Allan Bradley1,2
1Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
2Department of Medicine, University of Cambridge, Cambridge CB2 2QQ, UK
3Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0XY, UK
4Department of Haematology, University of Cambridge, Cambridge CB2 0PT, UK
5EMBL-EBI European Bioinformatics Institute, Hinxton, Cambridge CB10 1SD, UK
Recent advances in cancer immunology have shed light on the mechanisms that regulate the interaction of the immune system with cancer, along with the strategies of immune evasion that cancer cells employ in order to survive in the presence of anti-cancer immune responses. An accumulation of data in recent years has identified several immune checkpoint molecules, whose normal role is to down-regulate immune responses in order to maintain immune homeostasis. Expression of these molecules by cancer cells allows them to efficiently evade anti-cancer immune responses. The great potential of these findings is highlighted by the recent approval of therapeutic antibodies targeting and blocking immune checkpoint molecules. Examples include inhibitors of programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA4), for the treatment of various cancers. Development of novel immunotherapies constitutes an extremely promising clinical development. The advent of CRISPR/Cas9 genome editing technologies and their application in forward genetic screens has revolutionised our ability to interrogate the cancer genome and identify genetic vulnerabilities and essentialities. With these in mind, I am carrying out in vitro and in vivo CRISPR/Cas9 genome-wide forward genetic screens. These will, on one hand, enable the identification of genes whose knockouts sensitise cancer cells to the immune system. These genes would be ideal candidates as targets of immunotherapeutic strategies. On the other hand, the screens will also allow the characterisation of novel cancer immune evasion pathways. I anticipate that these studies will aid the identification of novel therapeutic cancer targets.
Streamlined production, application, and analysis of pooled genome-wide sgRNA lentiviral libraries
Cornelia Hampe1, Thomas P. Quinn2, Mei Fong2, Lily Lee2, Nathalie Bolduc2, Matthew H. Rowe2, Baz Smith2, Michael Haugwitz2, Andrew Farmer2
1Takara Bio Europe SAS, 78100 Saint-Germain-en-Laye, France
2Takara Bio USA, Inc., Mountain View, CA 94043, USA
Genome-wide loss-of-function genetic screens are a powerful way to identify novel protein functions and biological processes within a cell. Current methods using pooled sgRNAs in loss-of-function screens rely on lentiviral vector-based delivery followed by next-generation sequencing (NGS) to analyze the resulting distribution of sgRNA sequences in screened cell populations. Inherent challenges include maintaining sgRNA representation in lentiviral plasmids, achieving optimal titers upon scale-up of lentivirus production, and preparing high-quality NGS libraries that accurately reflect the distribution of sgRNA sequences. Here we present a streamlined approach for producing Cas9+/sgRNA+ cell populations in sufficient quantities for a genome-wide screen, and for generating NGS libraries used to assess changes in sgRNA representation, using the Guide-it CRISPR Genome-Wide sgRNA Library System. Our methods enable even novice users to perform genome-wide phenotypic screens without concerns for sgRNA representation, low virus titer, or NGS library preparation.
Marek’s disease virus-encoded miR-155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines
Yaoyao Zhang1, Na Tang1, 2, Jun Luo3, 4, Man Teng3, Katy Moffat1, Zhiqiang Shen2, Venugopal Nair1, 5, 6, Yongxiu Yao1
1The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey, United Kingdom GU24 0NF
2Binzhou Animal Science and Veterinary Medicine Academy & UK-China Centre of Excellence for Research on Avian Diseases, Binzhou 256600, Shandong, PR China
3Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, PR China
4College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, PR China.
5The Jenner Institute Laboratories, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, United Kingdom
6Department of Zoology, University of Oxford, 11a Mansfield Road, Oxford, United Kingdom
Marek’s disease virus 1 (MDV-1), a lymphotropic α-herpesvirus that induces T-cell lymphomas in chickens, serves as an excellent model to study herpesvirus-induced oncogenesis. Similar to oncogenic human γ-herpesviruses such as Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), MDV-1 encodes several microRNAs (miRNAs). These include MDV-miR-M4, a functional ortholog of the chicken miRNA gga-miR-155, expressed at very high levels in MDV-1-induced T-cell lymphomas and lymphoblastoid cell lines derived from them. We have previously shown that MDV-miR-M4 is essential for the induction of tumours. However, it is unclear whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype. MDV-1-transformed lymphoblastoid cell lines (LCLs) are widely used for interrogating the roles of viral and host genes in situ in MDV-induced lymphomas. Using the CRISPR/Cas9 editing approach, we have successfully deleted MDV-miR-M4 from the MDV genomes in the MDV-1-transformed cell line MDCC-HP8. The deletion of miR-M4 was confirmed by PCR, sequencing and qRT-PCR. Continued proliferation of the miR-M4-5p deleted clones of MDCC-HP8 cell lines demonstrated that miR-M4-5p expression is non-essential for maintaining the transformed phenotype of MDV-1-transformed LCLs. This is the first demonstration examining the direct role of an oncogenic miRNA in maintaining the transformation of the virus transformed cell line.
Genome Editing 2019 Sponsors
Gold Sponsor and Exhibitor
Bio-Rad Laboratoriesis a world leader in providing products for the life science research and diagnostic markets. In our Life Science Group, we build the industry leading solutions for oncology research, including the highly sensitive Droplet Digital™ PCR technology and our new technology for single-cell sequencing, the ddSEQ™ Single-Cell Isolator.
Gold Sponsor and Exhibitor
Merck is the preeminent life science company, supplying Sigma-Aldrich brand gene editing products and services. With over 300,000 products, including CRISPR reagents, whole genome screening libraries (including Sanger arrayed and GeCKO pooled libraries), validation services and technical expertise, Merck has committed to solving the toughest problems in life science.
Website: www.sigma-aldrich.com.
Bronze Sponsor and Exhibitor
Lonza provides the pharma market with the tools that life-science researchers use to develop and test therapeutics, beginning with basic research stages on to the final product release. Lonza’s bioscience products and services range from cell culture and discovery technologies for research to quality control tests and software that ensures product quality. Lonza Bioscience Solutions serves research customers worldwide in pharmaceutical, biopharmaceutical, biotechnology and personal care companies. The company delivers physiologically relevant cell biology solutions and complete solutions for rapid microbiology.
Lonza Cologne GmbH, Nattermannallee 1, Koeln, 50829, Germany
Phone: +49-221-99199-0, Fax: +49-221-99199-111
e-mail: scientific.support.eu@lonza.com
Bronze Sponsor and Exhibitor
Integrated DNA Technologies (IDT) is a leader in the manufacture and development of products for the research and diagnostic life science market. The largest supplier of custom nucleic acids in the world, IDT serves academic research, biotechnology, and pharmaceutical development communities.
IDT products support a wide variety of applications, including next generation sequencing (NGS), DNA amplification, SNP detection, microarray analysis, expression profiling, gene quantification, and synthetic biology. Platform-independent NGS products and services are available in addition to DNA and RNA oligonucleotides, qPCR assays, siRNA duplexes, and custom gene synthesis. Individually-synthesized xGen™ Lockdown™ Probes enable improved target capture. IDT also manufactures custom adaptors, fusion primers, Molecular Identifier tags (MIDs), and other workflow oligonucleotides for NGS. A TruGrade™ processing service is also available to reduce oligonucleotide crosstalk during multiplex NGS.
Serving over 80,000 life sciences researchers, IDT is widely recognized as the industry leader in custom oligonucleotide manufacture due to its unique capabilities. IDT pioneered the use of high throughput quality control (QC) methods and is the only oligonucleotide manufacturer to offer purity guarantees and 100% QC. Every oligonucleotide is analyzed by mass spectrometry and purified oligonucleotides receive further analysis by CE and HPLC. The company maintains an engineering division dedicated to advancing synthesis, processing technology, and automation. An in-house machine shop provides rapid prototyping and custom part design/control. Additionally, IDT offers unrivalled customer support, receiving approximately 100,000 calls annually with an average wait time of only 8 seconds.
A dedicated GMP manufacturing facility for molecular diagnostics provides oligonucleotides for In Vitro Diagnostic Devices (IVDs) or Analyte Specific Reagents (ASRs) for Laboratory-Developed Tests (LDTs). This manufacturing process is customer-defined and controlled, and facilitates progression from research to commercialization.
