7th International Conference & Exhibition

Welcome to The Conference

We are delighted to welcome you to our 7th annual genome editing conference, which was held virtually due the ongoing COVID19 pandemic, the need for social distancing and travel restrictions and featured:

• A high-impact, packed day of talks, discussions and on-demand breakout rooms
• Oral presentations on latest developments in the field of genome editing by an international faculty of researchers from academia and industry
• Updates on CRISPR patents
• Digital posters
• Virtual trade exhibition

Agenda

World Time Converter: https://www.worldtimebuddy.com

Presentation language: English

Tuesday 30th March 2021

1055: Welcome and housekeeping

Chair: Dr Lydia Teboul

1100: Professor Jose-Carlos Segovia, Head of Division, Hematopoietic Innovative Therapies Division
Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
CRISPR/Cas9 gene editing – from disease modelling to clinically applicable strategies

1120: 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
CRISPR/Cas9 Deletion of a Deep-Intronic Splicing Mutation Followed by NHEJ Repair in CFTR as a Potential Therapy for Cystic Fibrosis

1140: Dr Alessia Cavazza, Lecturer in Gene Therapy, UCL GOS Institute of Child Health, London, UK
Hematopoietic gene editing for the treatment of primary immunodeficiency diseases

1200: Dr Karen Keeshan, Reader and Principal Investigator, Paul O’Gorman Leukaemia Research Centre, University of Glasgow, Glasgow, UK
Generating leukaemia-inducing oncogene using CRISPR/Cas9 to model human disease

1220: Dr Lydia Teboul, Head of Molecular and Cellular Biology, The Mary Lyon Centre, MRC Harwell Institute, Harwell, UK
Long-read sequencing for the validation of alleles produced by genome editing

1240: Dr Ben Davies, Head, Transgenic Core Facility, Wellcome Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford UK
What to do when CRISPR/Cas9 is too efficient – strategies when addressing genes essential for viability or fertility

1300: Dr Peter Mouritzen, VP Market and Applications Development, Samplix, Denmark
Validation of Genome Editing through Indirect Sequence Capture and Long-Read Sequencing

1320: Break (break out rooms with the sponsors/digital stands)

Chair: Dr Ben Davies

1350: Dr Philip Webber, Partner at Dehns Oxford, Oxford, UK
CRISPR patent wars update

1410: Dr Liyang Zhang, Staff Scientist, Integrated DNA Technologies, Coralville, IA, USA
A sequence-to-function map of LbCas12a enabled rapid isolation of mutants with enhanced activity

1430: Professor Afaf El-Sagheer, Professor, Department of Chemistry, University of Oxford, Oxford, UK
Extending the Boundaries of Nucleic Acid Chemistry for therapeutics and Synthetic Biology

1450: Dr Leopard Parts, The Wellcome Trust Sanger Center, Hinxton, Cambridgeshire, UK
Biases in CRISPR/Cas9 editing outcomes

1510: Dr Gabrielle Wheway, Lecturer in Functional Genomics, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK
CRISPR-based high-content imaging assays

1530: Dr Qianxin Wu, Staff Scientist, The Wellcome Sanger Institute, Hinxton, Cambridgeshire, UK
Massively parallel characterisation of CRISPR activator efficacy in human induced pluripotent stem cells and neurons

1550: Dr Sumana Sharma, MRC Human Immunology Unit, University of Oxford, Oxford, UK
Applying genome-scale CRISPR-screening techniques to study cellular signalling events

1610: Dr Suzanne Snellenberg, Group Leader, OXGENE, Medawar Centre, Oxford, UK
Novel small molecule combinations that favour CRISPR/Cas-mediated homology-directed repair

1630: Close of meeting

Posters

A robust pipeline for high throughput CRISPR/Cas-mediated manufacture of engineered knock-out and knock-in cell lines

Hashtags: #GEOx21V, #DBlakemore

Daniel Blakemore, Simon Pollack, Victor van Gelder, Hind Ghezraoui, Sonia Moratinos, Jack Williams, Holly Prangley, Yves Du Toit, Claire Greenwood, Matt Burridge, Pela Derizioti, Ryan Cawood, Suzanne Snellenberg

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

The capacity to induce genetic modification in mammalian cells using CRISPR/Cas technology has the potential to revolutionise how we understand gene function. However, high-throughput application of this technology remains challenging, especially for complex alterations that rely on sporadic activity of the homology directed repair (HDR) pathway. In addition, cell types such as iPSCs are sensitive to these procedures, making it difficult to harness their potential. Here we present an automated, streamlined pipeline for generation of simple and complex gene modifications in multiple cell types including iPSCs. We first optimise transfection conditions, recovery from single cell sorting of the host cell lines and determine copy numbers of genes to be edited. In-house software, GRNADE and PRIMAPE, enables optimal sgRNA and primer design. We use ssODNs, the preferred template for HDR applications, for precise genome edits, and ascertain on-target cleavage and HDR efficiencies using in-house CRITIC software. We single cell sort transfected cells and use CellMetric® imaging software for automated and systematic monitoring to assure single cell clonality. Hamilton-robotics facilitate automated clone transfer for effective expansion. Clone screening for the correct edit is performed by CRITIC, followed by NGS analysis for validation. Using this pipeline, we have successfully generated knockouts and knock-ins in over 25 different cell lines, including iPSCs, for multiple applications from reagent verification and basic biology studies to complex disease modelling. We routinely achieve >95% editing efficiencies and up to 30% HDR efficiency using our optimised conditions. Also, the pipeline is optimised for multiple well-known iPSC culture systems, allowing us to meet demand for custom iPSC manufacture. Coupling CRISPR/Cas technology with high-throughput robotics facilitates scaling of simple complex genetic modifications, enabling large scale exploration of disease models.

Enhancing SaCas9 target specificity by rational directed mutagenesis

Hashtags: #GEOx21V, #HGhezraoui

Hind Ghezraoui, Gloria Mesa-Gil, Richard Parker-Manuel, Ryan Cawood, and Suzanne Snellenberg

OXGENE, Medawar Centre, Oxford Science Park Oxford, OX4 4HG, United Kingdom

Continued development of the CRISPR/Cas9 system is making therapeutic gene editing a viable biomedical tool. However, delivery of Streptococcus pyogenes (SpCas9) by adeno-associated viral vectors (AAV) is challenging due to the small packaging capacity of AAV (∼4.7 kb). This can be overcome by using the minimal SpCas9 ortholog Staphylococcus aureus Cas9 (SaCas9). Unfortunately, while many high-fidelity SpCas9 variants have been reported, far fewer SaCas9 variants have eliminated off-target editing, leaving off-target cleavage of unintended genomic sites a critical issue to be resolved for this ortholog. We developed a reporter plasmid that can be used to examine on- and off-target editing efficiency and used it to screen rationally engineered SaCas9 variants to identify those SaCas9 variants with enhanced specificity that could effectively discriminate a single base mismatch. We identified SaCas9 variants that dramatically reduced off-target effects, whilst maintaining robust on-target activity comparable to wild-type SaCas9. We confirmed editing at three genomic loci by CRITIC (CRISPR EdiTing InterferenCes) analysis using Sanger trace sequences. Thus, our variants could be used for genome editing applications where high fidelity is required.

Understanding the resistance landscape to gene drives targeting the doublesex gene in the malaria mosquito

Hashtags: #GEOx21V, #IMorianou

Ioanna Morianou¹, Andrew M. Hammond^1,2, Tony Nolan^1,3, Andrea Crisanti¹

¹Department of Life Sciences, Imperial College London, London, UK
²Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA
³Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK

Gene drives are selfish genetic elements with the potential to spread throughout entire insect populations for sustainable vector control. Recently, a CRISPR/Cas9-based gene drive was shown to eliminate caged populations of the malaria mosquito by targeting the female-specific exon of the highly conserved doublesex gene. However, the key question remains whether natural populations might be able to evolve resistance to the gene drive. Resistant alleles may be naturally occurring or generated by the drive itself. To investigate the potential for resistance at doublesex, we developed a high-throughput mutagenesis screen designed to enhance end-joining mutations at the gene drive target site. We discovered a few putatively resistant mutations that get generated at a very low frequency. This strategy can be used to evaluate gene drives for potential resistance prior to field testing. Informed by this assay, we developed a third generation gene drive design that can mitigate against known resistance and potentially eliminate malaria from entire regions in Africa.

Sponsor

Bronze Sponsor and Digital Exhibitor

Samplix provides researchers with tailored genomics tools to solve complex genetic landscapes. Delivered through the Xdrop^® platform, Samplix enables scientists to investigate in depth any genomic region of any species, enabling the enrichment of complex, including unknown, genomic regions. Xdrop allows researchers to isolate long (up to 100 kb) genomic regions from DNA samples, by knowing only a short portion (enough to design a short amplicon of 150 bp).

Main applications:

• Enrichment of long genomic regions
• Validation of genome editing (CRISPR, transgenic insertions, etc.)
• Identification of viral integration sites
• Distinguish genes from pseudogenes
• Gap-closing in draft genomes
• Phasing of variants
• Single cell whole genome amplification (< 6pg DNA)

Compatible with:

• Low input DNA (< 10 ng)
• Long read sequencing (Oxford nanopore or PacBio)
• Short-read sequencing (Illumina)
• Short tandem repeats
• Structural variation
• GC-rich regions

Samplix Xdrop workflow is available both as fully-supported platform (instrument, cartridges and reagents) as well as Service Provider.