TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
Abstract: Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15–30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation
- Location
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Deutsche Nationalbibliothek Frankfurt am Main
- Extent
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Online-Ressource
- Language
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Englisch
- Notes
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Nucleic acids research. - 42, 10 (2014) , 6762-6773, ISSN: 1362-4962
- Classification
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Biowissenschaften, Biologie
- Event
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Veröffentlichung
- (where)
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Freiburg
- (who)
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Universität
- (when)
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2019
- Creator
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Mussolino, Claudio
Alzuʾbi, Jamal
Fine, Eli J.
Morbitzer, Robert
Cradick, Thomas J.
Lahaye, Thomas
Bao, Gang
Cathomen, Anton
- DOI
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10.1093/nar/gku305
- URN
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urn:nbn:de:bsz:25-freidok-1227839
- Rights
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Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
- Last update
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25.03.2025, 1:41 PM CET
Data provider
Deutsche Nationalbibliothek. If you have any questions about the object, please contact the data provider.
Associated
- Mussolino, Claudio
- Alzuʾbi, Jamal
- Fine, Eli J.
- Morbitzer, Robert
- Cradick, Thomas J.
- Lahaye, Thomas
- Bao, Gang
- Cathomen, Anton
- Universität
Time of origin
- 2019
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Generation of safe CAR T cells to target B cell malignancies
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Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of [beta]-hemoglobinopathies
Establishing a zinc finger nuclease platform for targeted genome editing in human cell lines, primary keratinocyte stem cells and cardiomyocytes in vivo
Clinical genome editing to treat sickle cell disease - a brief update
Entwicklung von gentechnologischen Strategien zur Überwindung der Bluthirnschranke am Modell des Morbus Gaucher Typ 2
CRISPR-Cas9 based gene editing of the immune checkpoint NKG2A enhances NK cell mediated cytotoxicity against multiple myeloma
Hijacking-DNA -Repair (HDR)-CRISPR promotes seamless gene editing in human primary cells
Genome editing of SNAI1 gene in rhabdomyosarcoma: a novel model for studies of its role
Generation of safe CAR T cells to target B cell malignancies
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