Homologous gene targeting (HGT) is a precise but inefficient process for genome engineering. Several methods for increasing its efficiency have been developed, including the use of rare cutting endonucleases. However, there is still room for improvement, as even nuclease-induced HGT may vary in efficiency as a function of the nuclease, target site, and cell type considered. We have developed a high-throughput screening assay for the identification of factors stimulating meganuclease-induced HGT. We used this assay to explore a collection of siRNAs targeting 19,121 human genes. At the end of secondary screening, we had identified 64 genes for which knockdown affected nuclease-induced HGT. Two of the strongest candidates were characterized further. We showed that siRNAs directed against the ATF7IP gene, encoding a protein involved in chromatin remodeling, stimulated HGT by a factor of three to eight, at various loci and in different cell types. This method thus led to the identification of a number of genes, the manipulation of which might increase rates of targeted recombination. 1. Introduction The transfection of cells with exogenous DNA can be used to generate stable transformants with the exogenous sequence integrated into their genomes by random insertion (RI) or targeted integration driven by homologous recombination. In the case of homologous recombination, the integration of DNA into the genome is referred to as “homologous gene targeting” (HGT). Both RI and HGT depend on DNA double-strand break (DSB) repair mechanisms. DSBs are particularly hazardous events in cells. Two different and competing mechanisms repair DSBs. Homologous recombination (HR) involves the use of homologous sequences as a template for restoring genomic integrity upon DSB induction and is considered to be an error-free mechanism. Genetic and biochemical studies have shown that HR in yeast is mediated by the RAD52 epistasis group of genes [1], which are required to various extents for HGT. For example, almost no HGT is observed in the absence of a functional RAD52 gene, but substantial levels of recombination are observed in absence of RAD51 and RAD57 [2]. Homologs of these genes have been identified in vertebrates, including RAD51, RAD51B, RAD51C, RAD52, RAD54, XRCC2, and XRCC3, which have been shown to be necessary for HGT in the DT40 chicken lymphoid cell line [3–7]. In mouse embryonic stem (ES) cells, HGT is decreased slightly by mutations in BRCA2 [8], strongly by mutations in BRCA1 [9] and RAD54 [10] and is completely abolished by mutations in ERCC1/XPF [11]. By
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