Development and Optimization of CRISPR Prime Editing System in Photoautotrophic Cells.

Area of Science:

• Molecular Biology
• Plant Science
• Biotechnology

Background:

• Prime editing (PE) enables precise DNA modifications without double-strand breaks (DSB) or donor templates.
• Existing PE systems show limited efficiency in plant species, hindering their application.
• PE combines nickase Cas9 (nCas9) and reverse transcriptase (RT) with prime editing guide RNA (pegRNA).

Purpose of the Study:

• To develop an optimized prime editor (O-PE) for enhanced endogenous gene editing in Arabidopsis thaliana.
• To overcome the low editing efficiency of previous PE systems in plant cells.
• To provide a foundation for applying PE in other industrially relevant photoautotrophic cells.

Main Methods:

• Engineered an optimized prime editor (O-PE) for Arabidopsis thaliana.
• Tested alternative reverse transcriptases to improve editing efficiency.
• Analyzed the impact of different components on indel formation.

Main Results:

• Achieved an average editing efficiency of 1.15% in Arabidopsis thaliana cells, a 16.4-fold increase over previous reports.
• Identified that nCas9(H840A) fused to non-functional reverse transcriptase increases indels.
• Demonstrated the potential of O-PE for efficient plant gene editing.

Conclusions:

• The developed O-PE significantly improves gene editing efficiency in plant cells.
• This study offers a valuable tool for plant genetic engineering and functional genomics.
• The findings pave the way for applying PE in microalgae and other photoautotrophic organisms.