Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly
Open accessAbstract
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.
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Many studies have successfully disrupted CCR5 in various cell lines, primary T cells and hematopoietic stem cells, as well as in humanized mice.
“Since then, many studies have successfully disrupted CCR5 in various cell lines, primary T cells and hematopoietic stem cells (HSCs), as well as in humanized mice [30,31,32,38,39,40].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [40]
Kim et al. found that CCR5 ZFN induces off-target activity within the CCR2 gene even when there is a single mismatch between the targeted CCR5 sequence and CCR2 DNA.
“Kim et al. found that CCR5 ZFN induces off-target activity within CCR2 gene even when there is a single mismatch between the targeted CCR5 sequence and CCR2 DNA [40].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [40]
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- Authors
- Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., & Kim, J.-S.
- Affiliations
- Seoul National University (KR)
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2009-05-21
- Volume
- 19
- Issue
- 7
- Pages
- 1279-1288
- ISSN
- 1088-9051 (ISSN-L)
- PMC
- PMC2704428
- OpenAlex
- W2143208261
- Cited by
- 466
- References
- 53
- Citations by year
- 2026: 12 · 2025: 18 · 2024: 21 · 2023: 17 · 2022: 25 · 2021: 26 · 2020: 17 · 2019: 19 · 2018: 30 · 2017: 29 · 2016: 40 · 2015: 41 · 2014: 52 · 2013: 31 · 2012: 30
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genome Research (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Seoul National University Open Repository (Seoul National University) (submittedVersion)
- Scholarworks@UNIST (Ulsan National Institute of Science and Technology) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- zinc finger nucleases, targeted genome editing, modular assembly, CCR5 gene, HIV drug resistance, site-directed mutagenesis, gene therapy, T cell engineering, HIV infection
- MeSH terms
- Base Sequence, Endonucleases, Humans, Kidney, Luciferases, Molecular Sequence Data, Mutation, Sequence Homology, Nucleic Acid, Genome, Human, Zinc Fingers, Receptors, CCR5, DNA Breaks, Double-Stranded
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