Cargando…

T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-bindi...

Descripción completa

Detalles Bibliográficos
Autores principales: Rhiel, Manuel, Geiger, Kerstin, Andrieux, Geoffroy, Rositzka, Julia, Boerries, Melanie, Cathomen, Toni, Cornu, Tatjana I.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9986454/
https://www.ncbi.nlm.nih.gov/pubmed/36890979
http://dx.doi.org/10.3389/fgeed.2023.1130736
_version_ 1784901170291015680
author Rhiel, Manuel
Geiger, Kerstin
Andrieux, Geoffroy
Rositzka, Julia
Boerries, Melanie
Cathomen, Toni
Cornu, Tatjana I.
author_facet Rhiel, Manuel
Geiger, Kerstin
Andrieux, Geoffroy
Rositzka, Julia
Boerries, Melanie
Cathomen, Toni
Cornu, Tatjana I.
author_sort Rhiel, Manuel
collection PubMed
description Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.
format Online
Article
Text
id pubmed-9986454
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-99864542023-03-07 T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds Rhiel, Manuel Geiger, Kerstin Andrieux, Geoffroy Rositzka, Julia Boerries, Melanie Cathomen, Toni Cornu, Tatjana I. Front Genome Ed Genome Editing Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing. Frontiers Media S.A. 2023-02-20 /pmc/articles/PMC9986454/ /pubmed/36890979 http://dx.doi.org/10.3389/fgeed.2023.1130736 Text en Copyright © 2023 Rhiel, Geiger, Andrieux, Rositzka, Boerries, Cathomen and Cornu. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genome Editing
Rhiel, Manuel
Geiger, Kerstin
Andrieux, Geoffroy
Rositzka, Julia
Boerries, Melanie
Cathomen, Toni
Cornu, Tatjana I.
T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds
title T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds
title_full T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds
title_fullStr T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds
title_full_unstemmed T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds
title_short T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds
title_sort t-cast: an optimized cast-seq pipeline for talen confirms superior safety and efficacy of obligate-heterodimeric scaffolds
topic Genome Editing
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9986454/
https://www.ncbi.nlm.nih.gov/pubmed/36890979
http://dx.doi.org/10.3389/fgeed.2023.1130736
work_keys_str_mv AT rhielmanuel tcastanoptimizedcastseqpipelinefortalenconfirmssuperiorsafetyandefficacyofobligateheterodimericscaffolds
AT geigerkerstin tcastanoptimizedcastseqpipelinefortalenconfirmssuperiorsafetyandefficacyofobligateheterodimericscaffolds
AT andrieuxgeoffroy tcastanoptimizedcastseqpipelinefortalenconfirmssuperiorsafetyandefficacyofobligateheterodimericscaffolds
AT rositzkajulia tcastanoptimizedcastseqpipelinefortalenconfirmssuperiorsafetyandefficacyofobligateheterodimericscaffolds
AT boerriesmelanie tcastanoptimizedcastseqpipelinefortalenconfirmssuperiorsafetyandefficacyofobligateheterodimericscaffolds
AT cathomentoni tcastanoptimizedcastseqpipelinefortalenconfirmssuperiorsafetyandefficacyofobligateheterodimericscaffolds
AT cornutatjanai tcastanoptimizedcastseqpipelinefortalenconfirmssuperiorsafetyandefficacyofobligateheterodimericscaffolds