Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, Novosibirsk 630090, Russia.
Int J Mol Sci. 2022 Aug 3;23(15):8636. doi: 10.3390/ijms23158636.
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.
与使用传统遗传修饰方法通常实现的随机插入事件相比，将靶向 DNA 整合到基因组中的已知位置具有潜在优势。我们研究了携带靶向基因特异性整合的五个悬浮细胞系中植物和转基因 DNA 连接处的 DNA 重排的存在和程度。为了获得基因敲入，我们使用了两种模板，它们在基因组中与靶位点同源的侧翼 DNA 的存在与否上有所不同。对于靶向插入，我们选择了组蛋白 H3.3 基因中表达水平非常高的区域。我们的研究表明，所有获得的五个基因敲入细胞系在整合序列的边界处都有重排。在所有五个植物系中都发现了大约 100 个或更多 bp 来自右侧侧翼的显著重排。在五个系中的三个系中发现了来自左侧侧翼的超过 17 个 bp 的重组。对于基因敲入模板的两种变体（有侧翼和无侧翼）都检测到了重排，这表明侧翼的存在并不影响突变的发生。
