Genome editing employing the CRISPR/Cas9 system has been widely used and has become a promising tool for plant gene functional studies and crop improvement. However, most of the applied CRISPR/Cas9 systems targeting one locus using a sgRNA resulted in low genome editing efficiency. Here, we demonstrate the modification of the FAD2 gene in rice using a multiplex sgRNA-CRISPR/Cas9 genome editing system. To test the system’s efficiency for targeting multiple loci in rice, we designed two sgRNAs based on FAD2 gene sequence of the Oryza sativa Japonica rice. We then inserted the validated sgRNAs into a CRISPR/Cas9 basic vector to construct pYLCRISPRCas9PUbi-H:OsFAD2. The vector was then transformed into protoplast cells isolated from rice leaf tissue via PEG-mediated transfection, and rice calli using biolistic transformation. Direct DNA sequencing of PCR products revealed mutations consisting of deletions of the DNA region between the two target sgRNAs. The results suggested that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing system may be useful for crop improvement in monocot species that are recalcitrant to genetic modification, such as oil palm.

中文翻译：

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水稻FAD2基因的多重 CRISPR/Cas9 介导的基因组编辑：油棕模型基因组编辑系统

采用 CRISPR/Cas9 系统的基因组编辑已被广泛使用，并已成为植物基因功能研究和作物改良的有前途的工具。然而，大多数应用的 CRISPR/Cas9 系统使用 sgRNA 靶向一个基因座，导致基因组编辑效率低下。在这里，我们展示了使用多重 sgRNA-CRISPR/Cas9 基因组编辑系统对水稻中 FAD2 基因的修饰。为了测试系统靶向水稻中多个基因座的效率，我们设计了两种基于粳稻 FAD2 基因序列的 sgRNA。然后我们将经过验证的 sgRNA 插入到 CRISPR/Cas9 基本载体中以构建 pYLCRISPRCas9PUbi-H:OsFAD2。然后将载体通过 PEG 介导的转染转化为从稻叶组织中分离的原生质体细胞，并使用基因枪转化转化为水稻愈伤组织。PCR 产物的直接 DNA 测序揭示了由两个目标 sgRNA 之间 DNA 区域的缺失组成的突变。结果表明，多重 sgRNA-CRISPR/Cas9 基因组编辑系统的应用可能有助于油棕等难以接受基因改造的单子叶植物物种的作物改良。