Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, PqEB, Av W5 Norte Final 716, Brasília, DF, 70770-917, Brazil.
Department of Molecular Biology, University of Brasilia, Brasília, DF, 70910-900, Brazil.
Transgenic Res. 2021 Jun;30(3):239-249. doi: 10.1007/s11248-021-00246-x. Epub 2021 Apr 2.
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.
利用 CRISPR/Cas9 进行基因组编辑已被视为改良作物的有力工具。然而，其效率可以得到提高，特别是对于基因组复杂的作物，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，一种是单组件系统与双组件系统的比较。在简化系统中，单转录单元（STU），SpCas9 和 sgRNA 仅由一个启动子驱动，而在常规系统中，双组件转录单元（TCTU），SpCas9 由一个 pol II 启动子控制，sgRNAs 由一个 pol III 启动子控制。设计了一个三靶的多路复用系统，靶向两个不同的基因，GmIPK1 和 GmIPK2，编码植酸合成途径中的酶。这两种系统都使用毛状根大豆方法进行了测试。结果显示基因特异性编辑。对于 GmIPK1 基因，两种构型都观察到了编辑，缺失了 1 到 749 个碱基对；然而，TCTU 显示出更高的缺失频率。对于 GmIPK2，两种系统都观察到了主要缺失，但 STU 的编辑效率较低。这两种系统（STU 或 TCTU）都被证明能够在大豆中有效促进基因编辑。TCTU 构型被证明更可取，因为它更有效。STU 系统效率较低，但 CRISPR/Cas 盒的大小较小。
