Chimeric nucleases stimulate gene targeting in human cells
Abstract
Correction of gene defects in human somatic cells by targeting as has been used in murine embryonic stem cells (1, 2) has been precluded by the low spontaneous rate of gene targeting (3). However, creation of a DNA double-stranded break (DSB) in the genomic target (DSB-GT) can stimulate homologous recombination by over 1000-fold (4). \n \nWe can rapidly and quantitatively measure gene targeting by correcting a mutation in a green fluorescent protein (GFP) gene that has been stably integrated into the genome (5) (fig. S1). With an optimized GFP gene targeting system, the introduction of a DSB by I–Sce I (Sce) stimulated GT >40,000-fold and the absolute rate of gene targeting reached 3 to 5% (fig. S2). Such a system, however, depends on the prior introduction of a Sce binding site into the target gene and cannot be used for endogenous genes. Chimeric nucleases (CNs) have the potential to create sequence-specific DSBs (6). CNs—fusions between zinc finger binding DNA binding domains and the endonuclease domain of Fok I—can sitespecifically cleave naked DNA in vitro (6), extrachromosomal DNA in Xenopus oocytes (7), and chromosomal DNA in Drosophila (8). CNs work as dimers, and their efficiency depends on the spacing and orientation of the zinc finger binding sites with respect to the length of the amino acid linker between the DNA binding and endonuclease domains (7, 9). \n \nQQR is an artificial zinc finger DNA binding domain that recognizes the sequence 5′-GGGGAAGAA-3′ with nanomolar affinity (10). We modified QQR chimeric nucleases (QQR-CNs) (7, 9) (Fig. 1A) and tested whether they stimulated gene targeting (Fig. 1B). The background rate of gene targeting was 0.71 events per million transfected cells (fig. S1C). QQRL18-CN stimulated gene targeting 17-fold on target QQR6 and 260-fold on target QQR8 (Fig. 1B). QQRL0-CN did not stimulate gene targeting on target QQR8, but it was as efficient as Sce in stimulating gene targeting by over 2000-fold on target QQR6 (Fig. 1B). QQRL18-CN showed some preference for an 8–base pair (bp) spacing between binding sites whereas QQRL0-CN preferred 6-bp spacing. Thus, removing the linker between the zinc spacfinger and the nuclease domains increased the activity and specificity of the fusion protein in mammalian cells. As controls, we showed that the CNs did not stimulate gene targeting if (i) they lacked a nuclear localization signal, (ii) there was a single binding site rather than an inverted repeat binding site in the target, and (iii) the cognate binding site was changed. Thus, homodimers of CNs are potent stimulators of gene targeting in human somatic cells.
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If nuclease action mutates unintended targets that are important for cellular physiology, the treated cells may become dysfunctional or even die.
“For example, if the nuclease action mutates unintended targets that are important for cellular physiology, the treated cells may become dysfunctional or even die [61,62,63].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [62]
The fundamental safety concern for using ZFN mediated gene disruption for human therapy is the specificity of gene targeting.
“The fundamental safety concern for using ZFN mediated gene disruption for human therapy is the specificity of gene targeting.”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [62]
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- Authors
- Porteus, M. H., & Baltimore, D.
- Affiliations
- California Institute of Technology (US)
- Corresponding author
- Porteus, Matthew H.
- Baltimore, David
- Venue
- Science
- Published
- 2003-05-02
- Volume
- 300
- Issue
- 5620
- Pages
- 763-763
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- OpenAlex
- W2051395442
- Cited by
- 741
- References
- 10
- Citations by year
- 2026: 8 · 2025: 18 · 2024: 25 · 2023: 16 · 2022: 26 · 2021: 30 · 2020: 34 · 2019: 33 · 2018: 27 · 2017: 28 · 2016: 52 · 2015: 45 · 2014: 69 · 2013: 41 · 2012: 51
- FWCI
- 9.12
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Science (publishedVersion)
- PubMed (publishedVersion)
- CaltechAUTHORS (California Institute of Technology) (submittedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM039458, R01 GM39458)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- gene targeting, homologous recombination, zinc finger DNA-binding domain, DNA double-strand breaks, linker length, somatic cell therapy, gene therapy vectors, somatic cell hybrids, somatic cell nuclear transfer
- MeSH terms
- Cell Line, DNA, Humans, Luminescent Proteins, Mutation, Recombinant Fusion Proteins, Recombination, Genetic, Transfection, Deoxyribonucleases, Type II Site-Specific, Zinc Fingers, Gene Targeting, Dimerization, Nuclear Localization Signals, Saccharomyces cerevisiae Proteins, Green Fluorescent Proteins
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