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@ARTICLE{Rhiel:274186,
      author       = {M. Rhiel and K. Geiger and G. Andrieux$^*$ and J. Rositzka
                      and M. Börries$^*$ and T. Cathomen and T. I. Cornu},
      title        = {{T}-{CAST}: {A}n optimized {CAST}-{S}eq pipeline for
                      {TALEN} confirms superior safety and efficacy of
                      obligate-heterodimeric scaffolds.},
      journal      = {Frontiers in genome editing},
      volume       = {5},
      issn         = {2673-3439},
      address      = {Lausanne},
      publisher    = {Frontiers Media},
      reportid     = {DKFZ-2023-00481},
      pages        = {1130736},
      year         = {2023},
      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.},
      keywords     = {FokI (Other) / TALEN (Other) / chromosomal rearrangements
                      (Other) / chromosomal translocations (Other) / designer
                      nuclease (Other) / obligate heterodimer (Other) / off-target
                      effects (Other) / preclinical risk assessment (Other)},
      cin          = {FR01},
      ddc          = {630},
      cid          = {I:(DE-He78)FR01-20160331},
      pnm          = {899 - ohne Topic (POF4-899)},
      pid          = {G:(DE-HGF)POF4-899},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:36890979},
      pmc          = {pmc:PMC9986454},
      doi          = {10.3389/fgeed.2023.1130736},
      url          = {https://inrepo02.dkfz.de/record/274186},
}