Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.

中文翻译：

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T 细胞中多基因基因组编辑过程中 DNA 易位的检测和调节。

使用 DNA 核酸内切酶进行多重基因组编辑具有广泛的应用，包括用于细胞疗法，但染色体易位，即诱导基因组同时断裂的天然副产品，尚未得到详细探索。在这里，我们应用各种 CRISPR-Cas 核酸酶来编辑人类原代 T 细胞中的 T 细胞受体 alpha 和 beta 2 微球蛋白基因，并综合评估由此产生的易位的频率和稳定性。使用三种正交方法（液滴数字 PCR、单向测序和中期荧光原位）进行彻底的易位频率分析杂交）产生了可比较的结果，并且在两个同时进行的 CRISPR-Cas9 诱导编辑之间的总体易位率约为 7%。此外，我们表明，当使用不同的核酸酶组合时，或通过存在用于多重基因组编辑的同源单链寡核苷酸供体时，可以减少染色体易位。重要的是，减少易位的两种不同方法与细胞治疗应用兼容。