Rhiel Manuel, Geiger Kerstin, Andrieux Geoffroy, Rositzka Julia, Boerries Melanie, Cathomen Toni, Cornu Tatjana I
Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany.
Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, Freiburg, Germany.
Front Genome Ed. 2023 Feb 20;5:1130736. doi: 10.3389/fgeed.2023.1130736. eCollection 2023.
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 and 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.
转录激活样效应物核酸酶（TALENs）是已进入临床阶段的可编程核酸酶。二聚体的每个亚基都由一个DNA结合结构域组成，该结构域由一系列与FokI核酸内切酶的催化活性部分融合的TALE重复序列组成。当两个TALEN臂在近距离结合DNA时，FokI结构域二聚化并诱导产生交错末端的DNA双链断裂。在本研究中，我们描述了TALEN特异性CAST-Seq（T-CAST）的实施和验证，这是一种基于CAST-Seq的流程，可识别TALEN介导的脱靶效应，以高保真度指定脱靶位点，并预测导致脱靶切割的TALEN配对构象。我们通过评估设计用于靶向 和 位点的两种混杂TALEN的脱靶效应来验证T-CAST。这些TALEN的表达在原代T细胞中导致靶位点与各种脱靶位点之间的高水平易位。对FokI结构域进行氨基酸替换，使TALEN成为 obligate-heterodimeric（OH-TALEN），减轻了上述脱靶效应，且不损失靶向活性。我们的研究结果突出了T-CAST在评估TALEN设计核酸酶的脱靶效应和评估缓解策略方面的重要性，并提倡使用 obligate-heterodimeric TALEN支架进行治疗性基因组编辑。
