Cargando…

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

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 ef...

Descripción completa

Detalles Bibliográficos
Autores principales: Mussolino, Claudio, Alzubi, Jamal, Fine, Eli J., Morbitzer, Robert, Cradick, Thomas J., Lahaye, Thomas, Bao, Gang, Cathomen, Toni
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041469/
https://www.ncbi.nlm.nih.gov/pubmed/24792154
http://dx.doi.org/10.1093/nar/gku305
_version_ 1782318683935211520
author Mussolino, Claudio
Alzubi, Jamal
Fine, Eli J.
Morbitzer, Robert
Cradick, Thomas J.
Lahaye, Thomas
Bao, Gang
Cathomen, Toni
author_facet Mussolino, Claudio
Alzubi, Jamal
Fine, Eli J.
Morbitzer, Robert
Cradick, Thomas J.
Lahaye, Thomas
Bao, Gang
Cathomen, Toni
author_sort Mussolino, Claudio
collection PubMed
description 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.
format Online
Article
Text
id pubmed-4041469
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-40414692014-06-11 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 Cathomen, Toni Nucleic Acids Res Gene regulation, Chromatin and Epigenetics 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. Oxford University Press 2014-06-01 2014-05-03 /pmc/articles/PMC4041469/ /pubmed/24792154 http://dx.doi.org/10.1093/nar/gku305 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Gene regulation, Chromatin and Epigenetics
Mussolino, Claudio
Alzubi, Jamal
Fine, Eli J.
Morbitzer, Robert
Cradick, Thomas J.
Lahaye, Thomas
Bao, Gang
Cathomen, Toni
TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
title TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
title_full TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
title_fullStr TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
title_full_unstemmed TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
title_short TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
title_sort talens facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
topic Gene regulation, Chromatin and Epigenetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041469/
https://www.ncbi.nlm.nih.gov/pubmed/24792154
http://dx.doi.org/10.1093/nar/gku305
work_keys_str_mv AT mussolinoclaudio talensfacilitatetargetedgenomeeditinginhumancellswithhighspecificityandlowcytotoxicity
AT alzubijamal talensfacilitatetargetedgenomeeditinginhumancellswithhighspecificityandlowcytotoxicity
AT fineelij talensfacilitatetargetedgenomeeditinginhumancellswithhighspecificityandlowcytotoxicity
AT morbitzerrobert talensfacilitatetargetedgenomeeditinginhumancellswithhighspecificityandlowcytotoxicity
AT cradickthomasj talensfacilitatetargetedgenomeeditinginhumancellswithhighspecificityandlowcytotoxicity
AT lahayethomas talensfacilitatetargetedgenomeeditinginhumancellswithhighspecificityandlowcytotoxicity
AT baogang talensfacilitatetargetedgenomeeditinginhumancellswithhighspecificityandlowcytotoxicity
AT cathomentoni talensfacilitatetargetedgenomeeditinginhumancellswithhighspecificityandlowcytotoxicity