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.
Highlights
Targeted engineering of the human genome in somatic cells is a powerful means to study functional consequences of mutations found in the genomes of cancer cells or in patients with inherited genetic disorders, and potentially for gene therapy of these diseases
Whereas in case of knock-outs, not all the knockout clones generated in the same experiment using the same molecular scissor, are isogenic because the small deletions are generated through non-homologous end-joining (NHEJ) repair of double-strand breaks (DSBs) at the target site and are not of same size [15]
RAAV technology solely relies on homologous recombination (HR)-based insertion of gene targeting construct and results in a highly defined alteration throughout the clones
Summary
Targeted engineering of the human genome in somatic cells is a powerful means to study functional consequences of mutations found in the genomes of cancer cells or in patients with inherited genetic disorders, and potentially for gene therapy of these diseases. One class of such tools, encompassing the zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), homing endonucleases, triplex-forming oligonucleotides and Targetrons, are engineered molecular scissors that enable targeted genome editing at high efficiency [1,2,3,4]. These technologies constitute efficient tools for genome editing but may give rise to off-target editing
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