Wang Zhibo, Shea Zachary, Li Qi, Wang Kunru, Mills Kerri, Zhang Bo, Zhao Bingyu
School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, United States.
Front Plant Sci. 2023 Feb 20;14:1111683. doi: 10.3389/fpls.2023.1111683. eCollection 2023.
CRISPR/Cas9-based genome editing system is a powerful tool for plant genetic improvement. However, the variable efficiency of guide RNA(s) (gRNA) represents a key limiting factor that hampers the broad application of the CRISPR/Cas9 system in crop improvement. Here, we employed the -mediated transient assays to evaluate the effectiveness of gRNAs for editing genes in and soybean. We designed a facile screening system based on indels that can be introduced by CRISPR/Cas9-mediated gene editing. A gRNA binding sequence (23 nucleotides) was inserted into the open reading frame of yellow fluorescent protein (YFP) gene (gRNA-YFP), which disrupted the YFP reading frame and results in no fluorescent signal when it was expressed in plant cells. Transiently co-expression of Cas9 and a gRNA targeting the gRNA-YFP gene in plant cells could restore the YFP reading frame and recover the YFP signals. We evaluated five gRNAs targeting and soybean genes and confirmed the reliability of the gRNA screening system. The effective gRNAs targeting , , and had been used to generate transgenic plants and resulted in expected mutations on each gene. While a gRNA targeting was confirmed to be ineffective in transient assays. This gRNA indeed failed to trigger target gene mutations in stable transgenic plants. Thus, this new transient assay system can be used to validate the effectiveness of gRNAs before generating stable transgenic plants.
基于CRISPR/Cas9的基因组编辑系统是植物遗传改良的强大工具。然而，导向RNA（gRNA）效率的变异性是阻碍CRISPR/Cas9系统在作物改良中广泛应用的关键限制因素。在此，我们采用病毒介导的瞬时分析来评估gRNA在水稻和大豆中编辑基因的有效性。我们基于CRISPR/Cas9介导的基因编辑可引入的插入缺失设计了一个简便的筛选系统。将一个gRNA结合序列（23个核苷酸）插入黄色荧光蛋白（YFP）基因的开放阅读框（gRNA-YFP），这会破坏YFP阅读框，并导致其在植物细胞中表达时无荧光信号。在植物细胞中瞬时共表达Cas9和靶向gRNA-YFP基因的gRNA可恢复YFP阅读框并恢复YFP信号。我们评估了靶向水稻和大豆基因的5个gRNA，并证实了gRNA筛选系统的可靠性。靶向水稻OsALS、OsPDS和GmPDS的有效gRNA已用于生成转基因植物，并在每个基因上产生了预期的突变。而一个靶向水稻OsEPSPS的gRNA在瞬时分析中被证实无效。该gRNA在稳定转基因植物中确实未能引发靶基因突变。因此，这种新的瞬时分析系统可用于在生成稳定转基因植物之前验证gRNA的有效性。
