Hye Joo Kim1,2,3
, Hyung Joo Lee1,3
, Hyojin Kim1
, Seung Woo Cho1
and Jin-Soo Kim1,2,4
1Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-742, South Korea;
2ToolGen, Inc., Biotechnology Incubating Center, Seoul National University, Gwanak-gu, Seoul 151-724, South Korea
3 These authors contributed equally to this work.
Broad applications of zinc finger nuclease (ZFN) technology-which allows targeted genome editing-in research, medicine, and biotechnology are hampered by the lack of a convenient, rapid, and publicly available method for the synthesis of functional ZFNs. Here we describe an efficient and easy-to-practice modular-assembly method using publicly available zinc fingers to make ZFNs that can modify the DNA sequences of predetermined genomic sites in human cells. We synthesized and tested hundreds of ZFNs to target dozens of different sites in the human CCR5 gene-a co-receptor required for HIV infection-and found that many of these nucleases induced site-specific mutations in the CCR5 sequence. Because human cells that harbor CCR5 null mutations are functional and normal, these ZFNs might be used for (1) knocking out CCR5 to produce T-cells that are resistant to HIV infection in AIDS patients or (2) inserting therapeutic genes at “safe sites” in gene therapy applications.
4 Corresponding author.
[Supplemental material is available online at www.genome.org.]
Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.089417.108.
Received November 21, 2008.
Accepted March 18, 2009.