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
- Standort
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Deutsche Nationalbibliothek Frankfurt am Main
- Umfang
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Online-Ressource
- Sprache
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Englisch
- Anmerkungen
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Nucleic acids research. - 42, 10 (2014) , 6762-6773, ISSN: 1362-4962
- Klassifikation
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Biowissenschaften, Biologie
- Ereignis
-
Veröffentlichung
- (wo)
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Freiburg
- (wer)
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Universität
- (wann)
-
2019
- Urheber
-
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
- Rechteinformation
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Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
- Letzte Aktualisierung
-
25.03.2025, 13:41 MEZ
Datenpartner
Deutsche Nationalbibliothek. Bei Fragen zum Objekt wenden Sie sich bitte an den Datenpartner.
Beteiligte
- Mussolino, Claudio
- Alzuʾbi, Jamal
- Fine, Eli J.
- Morbitzer, Robert
- Cradick, Thomas J.
- Lahaye, Thomas
- Bao, Gang
- Cathomen, Anton
- Universität
Entstanden
- 2019
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