Ribozyme-processed guide RNA enhances virus-mediated plant genome editing

Area of Science:

• Plant molecular biology
• Gene editing technologies
• Virology

Background:

• Subgenomic promoters are used in virus-induced gene editing to express guide RNAs (gRNAs).
• The precise transcription start site for these promoters and its impact on gRNA structure are not well understood.
• Variable 5' end sequences in gRNAs can reduce the efficiency of gene editing enzymes like SpCas9.

Purpose of the Study:

• To investigate the impact of subgenomic promoter-driven gRNA transcription on gene editing.
• To optimize gRNA structure for enhanced SpCas9 cleavage activity and gene editing efficacy.
• To develop a more efficient virus-induced gene editing system for plants.

Main Methods:

• Analysis of gRNA sequences generated by subgenomic promoters to identify 5' end variations.
• Introduction of a hammerhead ribozyme sequence between the subgenomic promoter and gRNA coding region.
• Assessment of SpCas9 cleavage activity and gene editing efficiency in wild tobacco.

Main Results:

• Variable 5' end overhangs were observed in gRNAs transcribed from subgenomic promoters.
• These 5' end overhangs were found to decrease SpCas9 cleavage activity.
• Insertion of a hammerhead ribozyme resulted in precise 5' ends for gRNAs, significantly increasing gene editing efficacy in wild tobacco.

Conclusions:

• Hammerhead ribozyme incorporation effectively creates precise 5' ends for gRNAs in virus-induced gene editing systems.
• This optimization enhances SpCas9 activity and improves gene editing efficiency in plants.
• The developed system holds promise for high-efficiency gene editing applications in various plant species.