Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Biotechnol J. 2022 Jul;17(7):e2100189. doi: 10.1002/biot.202100189. Epub 2021 Jun 19.
In virus-induced gene-editing system, subgenomic promoters have been used to express guide RNAs (gRNAs). However, the transcription initiation site of the subgenomic promoters remains elusive. Here, we examined the sequence of gRNAs expressed by subgenomic promoters and found the variable length of overhangs at 5'-end of gRNAs. The overhangs at 5'-end of gRNA decrease the cleavage activity of SpCas9. To overcome this problem, we inserted hammerhead ribozyme between the subgenomic promoter and gRNA and confirmed that gRNAs with a precise 5'-end increase the editing efficacy in wild tobacco. This system will be widely used for editing target genes in plants with high efficiency.
在病毒诱导的基因编辑系统中，亚基因组启动子被用于表达向导 RNA（gRNA）。然而，亚基因组启动子的转录起始位点仍然难以确定。在这里，我们检查了亚基因组启动子表达的 gRNA 的序列，发现 gRNA 的 5'端有可变长度的突出端。gRNA 5'端的突出端降低了 SpCas9 的切割活性。为了解决这个问题，我们在亚基因组启动子和 gRNA 之间插入了锤头状核酶，证实了具有精确 5'端的 gRNA 提高了野生烟草中的编辑效率。该系统将广泛用于高效编辑植物中的靶基因。
