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 Should you wish your abstract not to be published, please notify us in writing at the time of abstract submission.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.
Knock-in and Correction of ALS-associated Mutations in Induced Pluripotent Stem Cells Using Footprint-Free Drug Selection
Jenny S Greig, Graham Cocks, Erin C Hedges, Chris E Shaw
Maurice Wohl Clinical Neuroscience Institute, King’s College London, Institute of Psychiatry, Psychology and Neuroscience. London, UK
In order to investigate the effects of specific ALS-associated mutations on disease-specific cell types, mutations in ARPP21 have been introduced into induced pluripotent stem cells (iPSCs) from a control line, and mutations in TARDBP have been corrected from patients with the disease. The genome editing was performed using CRISPR Cas9, introducing a drug selection cassette within a piggyBac transposon which is seamlessly removed after puromycin selection of edited clones. The edited lines can then be differentiated into various cell types such as motor neurons to model disease.
A fast and reliable method for detecting single base editing in clonal cell lines
Cornelia Hampe1, Montse Morell2, Tatiana Garachtchenko2, Patrick Martin2, Baz Smith2, Michael Haugwitz2, and Andrew Farmer2
1 Takara Bio Europe SAS, 78100 Saint-Germain-en-Laye, France
2 Takara Bio USA, Inc., Mountain View, CA 94043, USA
One of the most powerful applications of genome editing is the introduction of base changes in specific genomic sites that mimic single nucleotide polymorphisms (SNPs) related to human diseases or the introduction of stop codons to generate gene knockouts. However, screening a large number of clones to identify those containing the engineered base of interest is still a bottleneck, especially in the absence of a phenotypic readout. To address this need, we developed a SNP-detection method that allows quick screening of clones from a 96-well plate. The method is based on PCR amplification of the genomic target site, followed by an enzymatic assay with fluorescence readout. The overall workflow takes approximately four hours and any positive fluorescent signal is highly correlated with the successful introduction of the desired SNP. SNP detection using this method is independent of the engineered nucleotide substitution and the surrounding targeted genomic loci.
The in(del)s and outs of automated CRISPR-Cas9 genome editing: A robust CRISPR-Cas9 pipeline for high throughput mammalian genome editing
Jennifer A Harbottle, Daniel A Mestre, Kethan S Suvarna, Tom Payne, Ryan Cawood, Lee Spraggon
Oxford Genetics Ltd., The Oxford Science Park, Medawar 1, Oxford OX4 4GA, UK
CRISPR-Cas9 technology has revolutionised the field of genome editing and has enabled high efficiency mammalian cell engineering for a variety of downstream applications. The Gene Editing Technologies group at Oxford Genetics Ltd has developed an automated, streamlined pipeline for the generation of knock-out cell lines starting from the selection of efficient sgRNA through to NGS validation of edited clonal cell lines. Each step of the CRISPR-Cas9 workflow has been optimised and quality-assured for high throughput, large-scale demands, but is also flexible and adaptable to more specialised, small-scale projects. Characteristics of the host cell line are first established by qPCR to determine copy number of the target gene of interest, and Hamilton robotics-driven CellMetric® imaging software to ascertain growth kinetics. TIDE analysis of indel formation is performed to precisely determine the cleavage efficiency of the Cas9-sgRNA complex at the gene target site of interest. Once validated, the Cas9-sgRNA complex is delivered to the host cell line via an optimised delivery method that is suited to the cell type; this includes plasmid-based transfection, lentiviral transduction, and delivery of the nuclease as an active ribonucleoprotein complex. FACS enrichment of fluorescent protein-expressing cells from a transfected cell pool is used to maximise the frequency of targeted cells thereby decreasing the number of clones required for successful editing. Clone expansion and assurance of single cell clonality are monitored by the automated and systematic use of CellMetric® imaging software. Finally, clone screening for correctly targeted gene knock-out is performed by NGS genotyping and subsequent phenotype validation by Wes™ protein analysis. The Gene Editing Technologies platform at Oxford Genetics Ltd thus offers expertise and state-of-the-art resources and facilities for rapid high throughput and efficacious generation of CRISPR-Cas9 gene-edited clonal cell lines.
Targeted gene knockout of dUTPase using CRISPR/Cas9 genome editing technology leads to early embryonic lethality in mice
Hajnalka Laura Pálinkás1,2, Gergely Rácz1,3, Zoltán Gál4, Gergely Tihanyi1,3, Elen Gócza4, László Hiripi4, Beáta G. Vértessy1,2,3
1 Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
2 Doctoral School of Multidisciplinary Medical Science, University of Szeged, Szeged, Hungary
3 Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
4 Agricultural Biotechnology Institute, Department of Animal Biotechnology, Gödöllő, Hungary
Multiple knock-out mouse models can provide key novel insights into dynamics of genome integrity. The object of our studies, dUTPase, is a key enzyme in genome maintenance. It catalyzes the hydrolysis of dUTP into pyrophosphate and dUMP, supporting low cellular dUTP/dTTP ratio thus prevents genomic uracil accumulation. The molecular mechanism of thymine-less cell death, induced by the lack of dUTPase, is poorly understood although several routinely applied chemotherapeutic drugs in the clinic (fluoropyrimidines, methotrexate and its derivatives); interfere with the de novo thymidylate biosynthetic pathway. Previous reports showed that overexpression of dUTPase causes partial resistance against fluoropyrimidines, while its deficiency sensitizes tumor cells. Therefore a better knowledge of dUTPase role and function is particularly important in medicine. To investigate the physiological role of dUTPase in vivo, we established mice lacking dUTPase using CRISPR/Cas9-mediated genome engineering. So far two transgenic mouse models (dut +/-) were gained with 6 or 47 base pairs deletion in the coding gene of dUTPase. After crossing heterozygous mutants no viable homozygous pups were born indicating a lethal mutation. dUTPase homozygous mutant embryos were identified among blastocysts, which exhibited a normal appearance, but homozygous embryos were never found by E8.5, suggesting that mutant embryos die immediately after implantation. Single blastocysts are being undertook real-time PCR analysis to investigate potential maternal contribution of dUTPase transcripts at pre-implantation stages. We also performed Western blot studies on tissues derived from wild type and dut +/- heterozygote tissues in order to quantify dUTPase protein expression level and immunohistochemical staining on mouse sections to investigate cellular localization pattern. Our results establish for the first time that dUTPase is indispensable for proper post-implantation embryogenesis in mouse.
Comparison of three methods to measure successful knock down of EGFP in HEK293-EGFP cells using CRISPR/Cas9
Caroline F Peddle 1, Michelle E McClements 1, Robert E MacLaren 1,2
1 Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford
2 Oxford Eye Hospital, Oxford, United Kingdom
HEK293-EGFP cells were transfected with plasmids expressing Staphylococcus aureus Cas9 and a gRNA construct, and harvested 48 hours post-transfection. 6 x EGFP-targeting gRNAs and 2 x non-targeting control gRNAs were screened. The reduction in EGFP expression following CRISPR/Cas9-mediated gene disruption was measured via three methods: mean grey value of fluorescence microscopy images, fluorescence spectroscopy, and qPCR. The greatest detected reduction of EGFP varied between these methods of measurement, at 20.1 %, 35.2 %, and 50.0 % when using mean grey value, fluorescence spectroscopy, and qPCR, respectively (One-way ANOVA, n = 5). qPCR detected, on average, 25.3 % and 11.5 % greater knock down than mean grey value and fluorescence spectroscopy, using the same gRNAs. However, qPCR demonstrated the most variation between biological replicates. Only 4 of the 6 x EGFP-targeting gRNAs produced a significant reduction in EGFP expression compared to the untransfected control when using qPCR assessment, whereas mean grey value and fluorescence spectroscopy measurements of EGFP were significantly reduced with all 6 x EGFP-targeting gRNAs.