{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Rhiel M"],"funding":["Bundesministerium für Bildung und Forschung","Deutsche Forschungsgemeinschaft"],"pagination":["1130736"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC9986454"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["5"],"pubmed_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 <i>CCR5</i> and <i>TRAC</i> 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."],"journal":["Frontiers in genome editing"],"pubmed_title":["T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds."],"pmcid":["PMC9986454"],"funding_grant_id":["01ZZ 1801B 01ZZ 2015 01EK2205","CA311/4-1 SFB1160-Z02"],"pubmed_authors":["Geiger K","Boerries M","Cornu TI","Rhiel M","Rositzka J","Andrieux G","Cathomen T"],"additional_accession":[]},"is_claimable":false,"name":"T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds.","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 <i>CCR5</i> and <i>TRAC</i> 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.","dates":{"release":"2023-01-01T00:00:00Z","publication":"2023","modification":"2026-05-29T05:31:35.266Z","creation":"2025-02-18T23:44:49.296Z"},"accession":"S-EPMC9986454","cross_references":{"pubmed":["36890979"],"doi":["10.3389/fgeed.2023.1130736"]}}