The Mechanism of Gene Targeting in Human Somatic Cells

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Gene targeting is important for basic research and clinical applications. In the laboratory, gene targeting is used to knockout genes so that loss-of-function phenotypes can be assessed. In the clinic, gene targeting is the gold standard to which most gene therapy approaches aspire. One of the most promising tools for gene targeting in humans is recombinant adeno-associated virus (rAAV). The mechanism by which rAAV performs gene targeting has, however, remained obscure. Here, we surprisingly demonstrate that the normally single-stranded rAAV performs gene targeting via double-stranded intermediates, which are mechanistically indistinguishable from standard plasmid-mediated gene targeting. Moreover, we establish the double-strand break (DSB) repair model as the paradigm to describe human gene targeting, and delineate the dynamics of crossovers in this model. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted such that the chromosome becomes the “attacker” instead of the “attackee”. Finally, we confirm that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations advance our understanding of the mechanism of human gene targeting and should readily lend themselves to developing improvements to existing methodologies.

Vyšlo v časopise: The Mechanism of Gene Targeting in Human Somatic Cells. PLoS Genet 10(4): e32767. doi:10.1371/journal.pgen.1004251
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004251

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