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PLoS One. 2012;7(9):e45169. doi: 10.1371/journal.pone.0045169. Epub 2012 Sep 18.

Rescue the failed half-ZFN by a sensitive mammalian cell-based luciferase reporter system.

Author information

1
Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, People's Republic of China.

Abstract

ZFN technology is a powerful research tool and has been used for genome editing in cells lines, animals and plants. The generation of functional ZFNs for particular targets in mammalian genome is still challenging for an average research group. The modular-assembly method is relatively fast, easy-to-practice but has a high failure rate. Some recent studies suggested that a ZFP with low binding activity might be able to form a working ZFN pair with another binding active half-ZFP. In order to unveil the potential ZFP candidates among those with low binding activities, this paper established a highly sensitive mammalian cell-based transcriptional reporter system to assess the DNA binding activities of ZFPs by inserting multiple copies of ZFN target sequence fragment (TSF) of an interested gene (e. g., hPGRN or hVEGF). Our results showed that this system increased the screening sensitivity up to 50-fold and markedly amplified the differences in the binding activities between different ZFPs. We also found that the targeted chromosomal gene repair efficiency of each hPGRN or hVEGF ZFN pair was in proportion with the combination of the binding activities of the ZFL (Left zinc finger) and ZFR (Right zinc finger). A hPGRN ZFR with low binding ability was able to form a biological active ZFN if combined with a hPGRN ZFL with relatively high binding ability. Lastly, site-specific genome editing by hPGRN ZFNs generated by this system was confirmed by sequencing, and the PGRN knock-out cell line showed significantly decreased cell growth compared with the control. Our system will provide a valuable tool for further optimizing the nucleases with regard to specificity and cytotoxicity.

PMID:
23028823
PMCID:
PMC3445457
DOI:
10.1371/journal.pone.0045169
[Indexed for MEDLINE]
Free PMC Article

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