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

Agenda

Thursday 12th March 2020 | The Jarvis Doctorow Hall | St Edmund Hall | Oxford, UK

Session 1: Chair Dr Ben Davies

0950: Dr Dominic Owens, De Bruijn Lab, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
Microhomologies are prevalent at Cas9-induced larger deletions

1020: Dr Peter Mouritzen, Vice President Application and Market Development, Samplix ApS, Denmark
Xdrop™ – A New Microfluidics Technology for the Analysis of CRISPR Editing

1050: Dr Lydia Teboul, Head of Molecular and Cellular Biology , The Mary Lyon Centre, MRC Harwell Institute, Harwell, UK
Evolving the generation by genome editing of complex alleles and their validation

1120: Refreshment break

1150: Dr Daniel Ebner, Principal Investigator, Target Discovery Institute, Head of Scientific Operation, National Phenotypic Screening Centre – Oxford, Nuffield Department of Medicine, University of Oxford, Oxford, UK
Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages

1220: Professor Stephen Hart, Professor in Molecular Genetics & Deputy Head of Department, Genetics and Genomic Medicine Department, UCL Great Ormond Street Institute of Child Health, London, UK
Gene editing with CRISPR/Cas9 as a potential therapy for cystic fibrosis

1250: Lunch break

Session 2 (Via Skype): Chair Professor Stephen Hart

1340: Dr Ultan McDermott, Chief Scientist, Oncology, AstraZeneca Oncology R&D, CRUK Cambridge Institute, Cambridge, UK
The landscape of resistance pathways in BRAF mutant colon cancer revealed by functional genetic screens

1410: Dr Katharina Boroviak, Team Leader,Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, Hinxton, UK
Genome engineering using CRISPR/Cas9: what could go wrong?

1430: Dr Cornelia Hampe, Takara Bio Europe, France
A fast and reliable method for SNP and knockin screening

1450: Dr Johanna De Castro Arce, Horizon Discovery, Cambridge, UK
Application of CRISPR-Cas9 in clinically relevant cell models

1510: Dr Ashley Jacobi, Integrated DNA Technologies, Coralville, IA, USA
Improved methods for CRISPR HDR using modified DNA donors with optimized design

1530: Refreshment break

Session 3: Chair Dr Lydia Teboul

1600: Dr Philip Webber, Dehns Attorneys Oxford, Oxford, UK
CRISPR patent wars update

1620: Dr Ben Davies,Wellcome Centre for Human Genetics, Roosevelt Drive, Oxford UK
Reducing mosaicism in mice using destabilized Cas9

1640: Dr Lars Hanssen, MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford, UK
Interrogating the function of CTCF binding sites in vivo via CRISPR-Cas9 genome engineering

1700: Dr Sumana Sharma, European Bioinformatics Institute, Wellcome Genome Campus, Cambridgeshire, UK
Dissecting context dependent cancer signalling processes using CRISPR-based approaches

1720: Discussion and close

Posters

A PCR based method to create long, single-stranded DNA donors for gene knockin applications

Montse Morell¹, Hiroyuki Matsumoto¹, Ying Mao¹, Tatiana Garachtchenko¹, Cornelia Hampe², Thomas P. Quinn¹, Michael Haugwitz¹, Andrew Farmer¹

¹Takara Bio USA, 1290 Terra Bella Ave., Mountain View, CA 94043, USA

²Takara Bio Europe SAS, 34 rue de la Croix de Fer, 78100 Saint-Germain-en-Laye, France

DNA donors for CRISPR/Cas9 knockins can either be double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). However, dsDNA has the tendency to randomly integrate into the genome, and therefore is of very limited use for precise gene editing applications. ssDNA has a drastically reduced tendency to randomly integrate into the genome and mainly inserts into the site specifically targeted by the Cas9/sgRNA complex. In addition, ssDNA does not trigger a strong cytotoxic response after being delivered into cells, unlike dsDNA. Despite being the preferred choice for use as the DNA donor fragment for knockin applications, ssDNA can only be synthetically produced in an affordable and error-free manner up to a length of 200 base pairs. Above this length, ssDNA synthesis becomes very costly and error-prone. Here we report a simple method to produce long ssDNA of up to 5 kb based on a PCR reaction followed by enzymatic degradation of one of the strands. The ssDNA produced with this novel method can be electroporated in conjunction with recombinant Cas9 protein and in vitro transcribed sgRNA in knockin experiments targeting difficult to manipulate cells like hiPSCs.

Manipulation of gene expression using CRISPR-activation and CRISPR-inhibition systems in human induced pluripotent stem cells

Phalguni Rath¹, Adrià Dangla Valls², Marta Pérez Alcántara¹, Mark McCarthy¹, Noel Buckley² and Ben Davies¹

¹Wellcome Centre Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7BN, UK

²Department of Psychiatry, Medical Sciences Division, University of Oxford, Oxford OX3 7JX, UK

In addition to achieving site-specific deletions and insertion, CRISPR systems have been adapted to allow experimental control of gene expression. Catalytically inactivated Cas9 (dCas9) fused to transcriptional activation / inhibition machinery can be recruited to the endogenous promoter regions of target genes, allowing up (CRISPRa) and down (CRISPRi) regulation of gene expression. Such systems can be used to perturb gene expression in more subtle and controlled ways than conventional techniques and might provide a better means of modelling disease-associated gene expression changes and their functional consequences. We have engineered a control human induced pluripotent stem (iPS) cell line with the CRISPRa and CRISPRi machinery, by using Bxb1 integrase mediated cassette exchange to insert expression cassettes for the dCas9 transcriptional activators/inhibitors at the AAVS1 safe-harbour locus. We have optimized the promoter used to drive the expression of the machinery to ensure consistent and reliable expression of the CRISPRa/i machinery in both undifferentiated and differentiated cell types. Once activated by a Cre-recombinase switch, target gene expression within these stem cells can be manipulated by sgRNA transfection using optimized RNA transfection reagents. We demonstrate up- and down-regulation of several different target genes, at both mRNA and protein levels by simple delivery of single guide RNAs as the inducing agent and explore the kinetics of the gene expression change.  We are also exploring the functionality of these systems for achieving gene expression changes in differentiated cells such as neurons, cardiomyocytes, macrophages and pancreatic endocrine precursors. The engineered cell lines allow an alternative means of probing gene function using the iPS cell model system and could facilitate high-throughput screening approaches with sgRNA libraries.

An optimised CRISPR/Cas platform to allow enhanced precise genome editing in mammalian cells

Paul Guy¹, Victor van Gelder¹, Hind Ghezraoui¹, Stephanie Turner¹, Sonia Moratinos¹, Ryan Cawood¹, Suzanne Snellenberg¹

OXGENE, Medawar Centre, The Oxford Science Park, 1 Robert Robinson Ave, Oxford OX4 4HG, UK

CRISPR/Cas technology enables gene editing by introducing a wide variety of genomic alterations at specific locations. CRISPR/Cas induces DNA double-strand breaks (DSBs), which are mostly repaired by the error-prone non-homologous end-joining (NHEJ) pathway. This pathway can induce insertions and deletions (indels) leading to disruption of gene function. However, when CRISPR/Cas is supplied alongside a homologous donor DNA template, it can stimulate homology-directed repair (HDR) instead. This facilitates precise genome editing, which can be used to generate cell models for drug discovery and mode-of-action analyses. While CRISPR/Cas technology is widely accessible, generating high-quality, validated gene edited cell lines still represents a significant challenge. Here, we provide examples of such engineering approaches, and demonstrate how we have coupled this process to high-throughput robotics to facilitate scaling of these disease models. OXGENE’s methods include the delivery of repair templates via either adeno-associated virus (AAV) or up to 8kb single-stranded oligonucleotides (ssODNs). This not only enhances CRISPR/Cas-mediated HDR, but also the delivery of dual-gRNAs to allow for chromosomal deletions/rearrangements to happen. For each cell line, we optimise delivery of CRISPR/Cas components, and enrich targeted cells from the transfected pool using selectable markers. We have automated the process of clone expansion, and therefore assurance of single cell clonality, using CellMetric® imaging software. Finally, we use Next Generation Sequencing (NGS) genotyping, to screen clones for precise genome engineering and, depending of downstream applications, we validate subsequent phenotypes using the “suicide gene” ablation approach or by expression of a reporter-gene. OXGENE is further expanding these technologies, along with its CRISPR screening platform, to applications around induced pluripotent stem cells (iPSCs).

Genome Editing 2020 Sponsors

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

Synthego: The Genome Engineering Experts

Synthego is a leading provider of genome engineering solutions. Our automated, full-stack genome engineering platform enables broader access to CRISPR to accelerate basic scientific discovery, uncover cures for diseases and develop novel synthetic biology applications.

Synthego’s Engineered Cell products include Knockout Cell Pools and Clones for any target gene of interest, which are available in more than 700 human cell lines. We are the first and only provider who guarantees success for our engineered cell products which in a cell pool means a knockout of 50% or better. Engineered Cells also includes an Advanced Cells category, which provides access to sophisticated project-based knock-outs and knock-ins in human cell lines and primary cells as well as some other mammalian cell lines.

Synthego’s CRISPRevolution is the first product line to offer economical access to fully synthetic RNA for high fidelity editing and increased precision in genome engineering. To complete the portfolio Synthego developed the Multiguide concept which is also utilized in our arrayed CRISPR libraries for reliable functional knock-outs in combination with less off-targets. These libraries include the whole human genome, druggable, GPCRs, kinases, and immuno-oncology targets, among others.

Recently Synthego added precision CRISPR editing of induced pluripotent stem cells, including knockouts, SNVs, and tags as well as the production of customized GMP-grade synthetic guide RNA (sgRNA) used for gene-editing, extending the company’s ability to support the full spectrum of R&D in the next generation of cell and gene therapies.

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.

Bronze Sponsor and Exhibitor

Samplix – Despite the widespread use of DNA sequencing, challenges remain in the genomics
field. Many genomic regions present technological challenges, with the result that virtually all genomes contain “dark” and “unknown” regions. It is apparent that many of these dark and unknown regions contain disease-causing mutations that have remained inaccessible – until now. The Xdrop™ platform from Samplix transforms DNA sequencing by bringing this dark genome to light. Xdrop™ allows researchers to isolate long (up to 100 kb), intact genomic regions from DNA samples. Any selected sequence is isolated along with long flanking regions, allowing the DNA sequence to be analyzed in its full genomic context.

Target regions are isolated direct from genomic DNA as single molecules. After
unbiased amplification, the target DNA sequences are ready for analysis. With Xdrop™, using long- or short-read sequencing, researchers can now explore:

• CRISPR off-target effects
• Virus integration sites
• Repetitive sequence regions
• Genome gaps

Bronze Sponsor and Exhibitor

Horizon Discovery Group plc (LSE: HZD) (“Horizon”) is a world leader in gene editing and gene modulation technologies. Horizon designs and engineers cells using its translational genomics platform, a highly precise and flexible suite of DNA editing tools (rAAV, ZFN, CRISPR and Transposon) and, following the acquisition of Dharmacon, Inc., its functional genomics platform comprising gene knockdown (RNAi) and gene expression (cDNA, ORF) tools, for research and clinical applications that advance human health. Horizon’s platforms and capabilities enable researchers to alter almost any gene or modulate its function in human or mammalian cell-lines.

Horizon offers an extensive range of catalogue products and related research services to support a greater understanding of the function of genes across all species and the genetic drivers of human disease and the development of personalised molecular, cell and gene therapies. These have been adopted by over 10,000 academic, drug discovery, drug manufacturing and clinical diagnostics customers around the globe, as well as in the Company’s own R&D pipeline.

Horizon is headquartered in Cambridge, UK, and is listed on the London Stock Exchange’s AIM market under the ticker “HZD”

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.