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Computational and molecular tools for scalable rAAV mediated genome editing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. (MOLCAN)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (MOLCAN)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The rapid discovery of potential driver mutations through large scale mutational analyses of human cancers generates a need to characterize their cellular phenotypes. Among the techniques for genome editing, recombinant adeno-associated virus (rAAV) mediated gene targeting is particularly suited to knock-in of single nucleotide substitutions. However, the generation of gene targeting constructs and the targeting process is time consuming and labor-intense. To facilitate rAAV mediated gene targeting, we developed the first software and complementary automation friendly vector tools to generate optimized targeting constructs for editing human protein encoding genes. By computational approaches, rAAV constructs for editing ~72% of bases in protein-coding exons were designed. Similarly, ~81% of genes were predicted to be targetable by rAAV mediated knock-out. A Gateway based cloning system for facile generation of rAAV constructs suitable for robotic automation was developed and used in successful generation of targeting constructs. Together, these tools enable automated rAAV targeting construct design, generation as well as enrichment and expansion of targeted cells with desired integrations.

National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-235563OAI: oai:DiVA.org:uu-235563DiVA: diva2:761231
Available from: 2014-11-05 Created: 2014-11-05 Last updated: 2018-01-11
In thesis
1. Understanding Cancer Mutations by Genome Editing
Open this publication in new window or tab >>Understanding Cancer Mutations by Genome Editing
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mutational analyses of cancer genomes have identified novel candidate cancer genes with hitherto unknown function in cancer. To enable phenotyping of mutations in such genes, we have developed a scalable technology for gene knock-in and knock-out in human somatic cells based on recombination-mediated construct generation and a computational tool to design gene targeting constructs. Using this technology, we have generated somatic cell knock-outs of the putative cancer genes ZBED6 and DIP2C in human colorectal cancer cells. In ZBED6-/- cells complete loss of functional ZBED6 was validated and loss of ZBED6 induced the expression of IGF2. Whole transcriptome and ChIP-seq analyses revealed relative enrichment of ZBED6 binding sites at upregulated genes as compared to downregulated genes. The functional annotation of differentially expressed genes revealed enrichment of genes related to cell cycle and cell proliferation and the transcriptional modulator ZBED6 affected the cell growth and cell cycle of human colorectal cancer cells. In DIP2C-/-cells, transcriptome sequencing revealed 780 differentially expressed genes as compared to their parental cells including the tumour suppressor gene CDKN2A. The DIP2C regulated genes belonged to several cancer related processes such as angiogenesis, cell structure and motility. The DIP2C-/-cells were enlarged and grew slower than their parental cells. To be able to directly compare the phenotypes of mutant KRAS and BRAF in colorectal cancers, we have introduced a KRASG13D allele in RKO BRAFV600E/-/-/ cells. The expression of the mutant KRAS allele was confirmed and anchorage independent growth was restored in KRASG13D cells. The differentially expressed genes both in BRAF and KRAS mutant cells included ERBB, TGFB and histone modification pathways. Together, the isogenic model systems presented here can provide insights to known and novel cancer pathways and can be used for drug discovery.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 37 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1054
Keyword
Genome editing, rAAV, ZBED6, DIP2C, KRAS, BRAF, colorectal cancer, tumor driver genes, cancer pathways
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-235680 (URN)978-91-554-9106-2 (ISBN)
Public defence
2014-12-19, Rudbeck Salen, Uppsala University, Rudbeck Laboratory SE-751 85, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-11-27 Created: 2014-11-07 Last updated: 2018-01-11

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CiteExportLink to record
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Citation style
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