DOI: 10.1002/biot.202100189

Abstract

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.

Publication Types

Journal Article

Keywords

CRISPR-Cas9 hammerhead ribozyme plant genome editing tobacco rattle virus vector virus-induced genome editing

Grant Support

• National Research Foundation of Korea (): 2018R1A5A7025409
• National Research Foundation of Korea (): 2021R1A2C4002413
• Institute for Basic Science (): R021-D1
• Rural Development Administration (): PJ01481603

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