title: Computational and molecular tools for scalable rAAV-mediated genome editing.
abstract: 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 ~71% 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.
authors.affiliation: Science For Life Laboratory, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
authors.forename: Ivaylo
authors.initials: I
authors.lastname: Stoimenov
authors.affiliation: Science For Life Laboratory, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
authors.forename: Muhammad Akhtar
authors.initials: MA
authors.lastname: Ali
authors.affiliation: Science For Life Laboratory, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Pandzic
authors.affiliation: Science For Life Laboratory, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden tobias.sjoblom@igp.uu.se.
authors.forename: Tobias
authors.initials: T
authors.lastname: Sjöblom
mesh_terms: D003198:Computer Simulation; D030541:Databases, Genetic; D000229:Dependovirus / Q000235:genetics*; D005786:Gene Expression Regulation; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D011995:Recombination, Genetic / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: D049452:Green Fluorescent Proteins