Identification of Genes Regulating Gene Targeting by a High-Throughput Screening Approach
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 effi...
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| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2011-01-01
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| Series: | Journal of Nucleic Acids |
| Online Access: | http://dx.doi.org/10.4061/2011/947212 |
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| Summary: | 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. |
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| ISSN: | 2090-021X |