Evaluating the effect of chromosomal context on zinc finger nuclease efficiency
© Bahr et al.; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
Zinc Finger Nuclease (ZFN) technology has provided researchers with a tool for integrating exogenous sequences into most cell lines or genomes in a precise manner. Using current methods, the efficiency of targeted integration (TI) into the host genome is generally low and is highly dependent on the ZFN activity at the genomic locus of interest. It is unknown if the ZFN binding and cutting efficiency is more dependent on the nucleotide recognition sequence or the chromosomal context in which the sequence is located.
We have taken a highly efficient ZFN pair (hAAVS1) from human studies and introduced the exogenous DNA sequence into the Chinese Hamster Ovary (CHO) genome in an attempt to improve the efficiency of targeted integration. A "Landing Pad" comprised of human AAVS1 sequence has been integrated into the CHO genome at 3 separate loci to determine if the ZFN's will work across species and if the cutting efficiency is affected by chromosomal context. The results of this study will help us to improve the overall efficiency of TI by using Landing Pads, particularly for genomic targets in which suitable ZFN's may not be available.
Comparing ZFN activity in CHO before and after Landing Pad Integration
ZFN Activity at Endogenous CHO Locus
ZFN Activity at Integrated Landing Pad
These results indicate that the chromosomal context of the ZFN recognition sequence has an effect on cutting efficiency. This study shows that TI can be performed with Landing Pads across species with high efficiency and provide researchers with additional tools for cell line engineering. Further development of Landing Pads could create highly engineered and multi-functional platforms that would facilitate more efficient and more tailored CHO cell modifications.
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