Genome editing using CRISPR/Cas9 has been highlighted as a powerful tool for crop improvement. Nevertheless, its efficiency can be improved, especially for crops with a complex genome, such as soybean. In this work, using the CRISPR/Cas9 technology we evaluated two CRISPR systems, a one-component vs. a two-component strategy. In a simplified system, the single transcriptional unit (STU), SpCas9 and sgRNA are driven by only one promoter, and in the conventional system, the two-component transcriptional unit (TCTU), SpCas9, is under the control of a pol II promoter and the sgRNAs are under the control of a pol III promoter. A multiplex system with three targets was designed targeting two different genes, GmIPK1 and GmIPK2, coding for enzymes from the phytic acid synthesis pathway. Both systems were tested using the hairy root soybean methodology. Results showed gene-specific edition. For the GmIPK1 gene, edition was observed in both configurations, with a deletion of 1 to 749 base pairs; however, the TCTU showed higher indel frequencies. For GmIPK2 major exclusions were observed in both systems, but the editing efficiency was low for STU. Both systems (STU or TCTU) have been shown to be capable of promoting effective gene editing in soybean. The TCTU configuration proved to be preferable, since it was more efficient. The STU system was less efficient, but the size of the CRISPR/Cas cassette was smaller.

使用 CRISPR/Cas9 进行基因组编辑已被强调为作物改良的强大工具。然而，它的效率可以提高，特别是对于具有复杂基因组的作物，如大豆。在这项工作中，我们使用 CRISPR/Cas9 技术评估了两种 CRISPR 系统，一种是单组分策略，另一种是双组分策略。在简化的系统中，单个转录单元 (STU) SpCas9和 sgRNA 仅由一个启动子驱动，而在常规系统中，双组分转录单元 (TCTU) SpCas9受 pol II 启动子控制并且 sgRNA 受 pol III 启动子的控制。针对两个不同的基因GmIPK1和GmIPK2设计了具有三个目标的多重系统，编码来自植酸合成途径的酶。两种系统都使用毛根大豆方法进行了测试。结果显示基因特异性版本。对于GmIPK1基因，在两种配置中都观察到了版本，删除了 1 到 749 个碱基对；然而，TCTU 显示出更高的 indel 频率。对于GmIPK2在两个系统中观察到主要的排除，但编辑效率低的STU。两种系统（STU 或 TCTU）都已被证明能够促进大豆中的有效基因编辑。事实证明，TCTU 配置更可取，因为它更有效。STU 系统效率较低，但 CRISPR/Cas 盒的尺寸较小。