TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity

Mussolino, Claudio; Alzubi, Jamal; Fine, Eli J.; Morbitzer, Robert; Cradick, Thomas J.; Lahaye, Thomas; Bao, Gang und Cathomen, Toni (2014): TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. In: Nucleic Acids Research, Bd. 42, Nr. 10: S. 6762-6773

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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Biologie > Department Biologie I
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
• ISSN: 0305-1048
• Sprache: Englisch