The Mechanism of Gene Targeting in Human Somatic Cells

Yinan Kan,Brian Ruis,Eric A Hendrickson,Sherry Lin,Sue Jinks-Robertson

doi:10.1371/journal.pgen.1004251

Abstract

Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. 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 to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

Highlights

Gene targeting is the process of intentionally altering a genetic locus in a living cell [1]
One of the most promising tools for gene targeting in humans is recombinant adeno-associated virus
We demonstrate that when a meganuclease is used to introduce a chromosomal double-strand break (DSB) to augment gene targeting, the mechanism of gene targeting is inverted such that the chromosome becomes the ‘‘attacker’’ instead of the ‘‘attackee’’

Summary

Introduction

Gene targeting is the process of intentionally altering a genetic locus in a living cell [1]. This technology has at least two applications of significant importance. The second application is the inactivation of genes (‘‘knockouts’’), a process in which the two wild-type alleles of a gene are disrupted to determine the loss-offunction phenotype associated with that particular gene. These two processes are conceptually reciprocal opposites of each other, they are mechanistically identical because both require a form of DNA double-strand break (DSB) repair (DSBR) termed homologous recombination (HR). A recombination intermediate is generated with two Holliday Junctions (HJs) that is identical to the intermediate of plasmidbased gene targeting that has been well-defined in yeast [2,5,6,7]

Results

Discussion

Conclusion