Guidelines for Poster Preparation
Please prepare your poster in A1 portrait format (59cm wide x 84cm long). Do not laminate your poster or use heavy printing material. Further information about poster sizes can be found on the following link:
Posters larger than A1 will only be displayed subject to the availability of space.
Maximum capacity 10 A1 potrait posters
Please ensure you have appropriate permissions for the publication of your abstract from the original copyright holders. Should you wish your abstract not to be published, please notify us in writing at the time of abstract submission.
Posters will be displayed for the full duration of the conference.Titles of accepted poster abstracts will be displayed below.
(Presenters in Bold)
If your abstract has been accepted for presentation but it does not appear in the list below, please let us know as soon as possible by email on CRISPR@LPMHealthcare.com.
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).