Use of internally nuclease-protected single-strand DNA oligonucleotides and silencing of the mismatch repair protein, MSH2,enhances the replication of corrected cells following gene editing
Use of internally nuclease-protected single-strand DNA oligonucleotides and silencing of the mismatch repair protein, MSH2,enhances the replication of corrected cells following gene editing
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DOI：
10.1002/jgm.1296
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发表时间：
2009-03-01
影响因子：
3.5
通讯作者：
Owen, James S.
中科院分区：
文献类型：
--
作者：
Papaioannou, Ioannis;Disterer, Petra;Owen, James S.
基因编辑是一种利用短单链DNA寡核苷酸（ssODNs）引入遗传变化的潜在强大技术。然而，错配修复系统降低了它们的效率，尤其是MSH2，它可能抑制基因编辑，尽管结果取决于所使用的寡核苷酸的读数和类型。此外，据报道，成功编辑的细胞在S期或g2期被阻滞。在本研究中，我们评估了一种新的ssODN设计和MSH2表达的下调是否允许复制基因编辑细胞的分离。方法用不同设计的ssodn靶向表达增强绿色荧光蛋白突变的中国仓鼠卵巢细胞，这些ssodn都能恢复荧光，从而可以通过流式细胞术监测校正事件。通过细胞分选分离转化细胞并培养以确定集落形成效率。小干扰RNA抑制MSH2表达，碘化丙啶或DRAQ5染色后，流式细胞术定量检测转染ssODN细胞的细胞周期分布。结果虽然硫代酸对ssODN末端进行保护的效率更高，但编辑后的细胞形成集落的潜力低于未修饰的ssODN。我们证实ssODN转染本身会扰乱细胞周期，而MSH2基因沉默会提高校正效率。然而，在这两种情况下，效果取决于受保护核苷酸的位置。重要的是，当内部保护ssODN与MSH2抑制联合使用时，转染后48-64小时观察到更高比例的g1期校正细胞。使用内部保护的ssODN和下调细胞MSH2活性可能有助于分离活的、积极复制的基因编辑细胞。版权所有John Wiley & Sons， Ltd. 2009
Background Gene editing is potentially a powerful technology for introducing genetic changes by using short single-stranded DNA oligonucleotides (ssODNs). However, their efficiency is reduced by the mismatch repair system, especially MSH2, which may suppress gene editing, although findings vary depending on readout and type of oligonucleotide used. Additionally, successfully edited cells are reported to arrest at the S- or G2-phase. in the present study, we evaluate whether a. novel ssODN design and down-regulation of MSH2 expression allows the isolation of replicating gene-edited cells.Methods Cultured Chinese hamster ovary cells expressing mutated enhanced green fluorescent protein were targeted with ssODNs of varying design, all capable of restoring fluorescence, which allows the monitoring of correction events by flow cytometry. Converted cells were isolated by cell sorting and grown to determine colony formation efficiencies. MSH2 expression was suppressed with small interfering RNA and the cell cycle distribution of cells transfected with ssODN was quantified by flow cytometry, following propidium iodide or DRAQ5 staining.Results Although efficiency was higher using ssODN end-protected with phosphorothioate, the potential of edited cells to form colonies was lower than those targeted with unmodified ssODN. We established that ssODN transfection itself perturbs the cell cycle and that MSH2 gene silencing increases correction efficiency. in both cases, however, the effect was dependent on the positioning of the protected nucleotides. Importantly, when internally protected ssODN was used in combination with MSH2 suppression, a higher proportion of G1-phase corrected cells was observed 48-64 h after transfection.Conclusions Use of internally protected ssODN and downregulating cellular MSH2 activity may facilitate isolation of viable, actively replicating gene-edited cells. Copyright (C) 2009 John Wiley & Sons, Ltd.