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

Abstract: 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

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Frontiers in genome editing. - 5 (2023) , 1130736, ISSN: 2673-3439

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2023
• Creator: Rhiel, Manuel ; Geiger, Kerstin ; Geoffroy, Andrieux ; Rositzka, Julia ; Börries, Melanie ; Cathomen, Anton ; Cornu, Tatjana I.

Further information

• DOI: 10.3389/fgeed.2023.1130736
• URN: urn:nbn:de:bsz:25-freidok-2342335
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 25.03.2025, 1:51 PM CET