BACKGROUND As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results. RESULTS In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission. CONCLUSIONS Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.

中文翻译：

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一系列CRISPR / Cas9大豆品系中突变和转基因的整合，丰度和传递。

背景技术与许多植物物种一样，大豆中的当前基因组编辑策略是通过将编码工程核酸酶的基因稳定地转化为基因组而启动的。转基因的表达导致在靶基因座处的双链断裂和修复，常常导致在预期位点的突变。由于大豆是具有20个染色体对的自花授粉物种，因此通常预期T0植物中的转基因与目标突变不相关，并且转基因/突变应独立分类。进入T1代，导致分离家族中转基因存在/缺失和等位基因状态的孟德尔组合。但是，这种预测并不总是与观察到的结果一致。结果在这项研究中，我们研究了大豆分离家族中三个不同的CRISPR / Cas9转基因之间的遗传模式及其各自的诱导突变。对四株T0植物和四株T1后代植物进行下一代重测序，然后对隔离家族进行更广泛的评估，揭示了不同谱系之间的预期遗传模式和意外遗传模式。这些意外的模式包括：（1）一个家族，其中T0转基因和突变没有传播给后代；（2）具有四个未连接的转基因插入的家族，其中两个分别位于旁源CRISPR靶标断裂位点；（3）观察到并传播突变但没有转基因整合或传播迹象的家庭。结论基因组重测序可提供高分辨率的转基因整合结构和基因编辑事件。这些事件的隔离模式可能会由于多种潜在机制而变得复杂。这包括但不限于植物嵌合体，多个未连接的转基因整合，有意和旁系靶标的编辑，转基因整合与靶位点之间的连接，以及在不将转基因整合到宿主基因组中的编辑试剂的瞬时表达。