Zelensky Alex N, Schimmel Joost, Kool Hanneke, Kanaar Roland, Tijsterman Marcel
Department of Molecular Genetics, Cancer Genomics Netherlands, Erasmus University Medical Centre, Rotterdam,, 3000 CA, The Netherlands.
Department of Human Genetics, Leiden University Medical Centre, PO Box 9600, Leiden,, 2300 RC, The Netherlands.
Nat Commun. 2017 Jul 7;8(1):66. doi: 10.1038/s41467-017-00124-3.
Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.
外源DNA的脱靶或随机整合阻碍了精确的基因组工程，并在临床基因治疗策略中带来安全风险。几十年来一直缺乏对随机整合的遗传学定义。在此，我们表明A家族DNA聚合酶θ（Pol θ）促进随机整合，而经典的非同源DNA末端连接起次要作用；聚合酶θ和经典非同源DNA末端连接双缺陷的细胞没有任何整合事件，这表明这两种机制定义了随机整合。相比之下，这些细胞中的同源重组并未减少，基因靶向效率提高到了100%。整合结果从主要是随机整合完全转变为仅基因靶向，为提高DNA递送和基因校正方法的疗效和安全性提供了一条合理的前进道路。随机脱靶整合事件会损害精确的基因靶向，并给基因治疗带来安全风险。在此，作者表明抑制聚合酶θ和经典非同源重组可消除随机整合。
Zotero 插件
