Computational and molecular tools for scalable rAAV-mediated genome editing
2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 5, e30Article in journal (Refereed) Published
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 suited for knock-in of single nucleotide substitutions and to a lesser degree for gene knock-outs. 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 similar to 71% of bases in protein-coding exons were designed. Similarly, similar to 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.
Place, publisher, year, edition, pages
2015. Vol. 43, no 5, e30
Cancer and Oncology
IdentifiersURN: urn:nbn:se:uu:diva-252714DOI: 10.1093/nar/gku1286ISI: 000352487100003PubMedID: 25488813OAI: oai:DiVA.org:uu-252714DiVA: diva2:811302