Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases
Open accessAbstract
Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The high specificity and affinity of these chimeric enzymes are based on custom-designed zinc finger proteins (ZFPs). To improve the performance of existing ZFN technology, we developed an in vivo evolution-based approach to improve the efficacy of the FokI cleavage domain (FCD). After multiple rounds of cycling mutagenesis and DNA shuffling, a more efficient nuclease variant (Sharkey) was generated. In vivo analyses indicated that Sharkey is >15-fold more active than wild-type FCD on a diverse panel of cleavage sites. Further, a mammalian cell-based assay showed a three to sixfold improvement in targeted mutagenesis for ZFNs containing derivatives of the Sharkey cleavage domain. We also identified mutations that impart sequence specificity to the FCD that might be utilized in future studies to further refine ZFNs through cooperative specificity. In addition, Sharkey was observed to enhance the cleavage profiles of previously published and newly selected heterodimer ZFN architectures. This enhanced and highly efficient cleavage domain will aid in a variety of ZFN applications in medicine and biology.
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Because the genome contains numerous sequence repetitions or highly homologous DNA sequences, efforts have been made to improve off-target effects and specificity of ZFN and TALEN enzymes.
“Because the genome contains numerous sequence repetitions or has highly homologous DNA sequences, efforts have been made in the last years to improve the offtarget effects and the specificity of these enzymes (Miller et al., 2007;Szczepek et al., 2007;Guo et al., 2010;Doyon et al., 2011).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Guo et al., 2010
The Discussion says that improving the FokI cleavage domain’s discrimination between highly homologous target sites may increase ZFN specificity and reduce off-target cleavage. It does not address TALENs or establish that numerous genomic repetitions motivated these efforts; the specificity and off-target benefits are framed as possibilities, not demonstrated outcomes. (LLM, conf=0.85)
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- Authors
- Guo, J., Gaj, T., & Barbas, C. F.
- Affiliations
- Scripps Research Institute (US)
- Corresponding author
- Barbas, Carlos F.
- ORCID
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2010-07
- Volume
- 400
- Issue
- 1
- Pages
- 96-107
- ISSN
- 0022-2836 (ISSN-L)
- PMC
- PMC2885538
- OpenAlex
- W2144141440
- Cited by
- 206
- References
- 40
- Citations by year
- 2026: 2 · 2025: 8 · 2024: 5 · 2023: 10 · 2022: 5 · 2021: 10 · 2020: 6 · 2019: 6 · 2018: 10 · 2017: 12 · 2016: 13 · 2015: 19 · 2014: 30 · 2013: 21 · 2012: 25
- FWCI
- 9.13
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health
- National Institute of General Medical Sciences (R01GM065059)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- zinc finger nucleases, directed evolution, in vivo evolution, DNA shuffling, targeted mutagenesis, sequence specificity, genome editing
- MeSH terms
- Animals, Cell Line, Humans, Models, Molecular, Molecular Sequence Data, Mutation, Protein Binding, Protein Conformation, Recombinant Fusion Proteins, Deoxyribonucleases, Type II Site-Specific, Zinc Fingers, Directed Molecular Evolution, Catalytic Domain
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