Programmable RNA N⁶-methyladenosine editing with CRISPR/dCas13a in plants

Area of Science:

• Molecular Biology
• Plant Science
• Epigenetics

Background:

• N⁶-methyladenosine (m⁶A) is a crucial RNA modification impacting mRNA processing and metabolism.
• Understanding individual m⁶A functions in plants requires tools for targeted modification and analysis.

Purpose of the Study:

• To create programmable tools for editing specific m⁶A sites on plant mRNAs.
• To investigate the functional and phenotypic consequences of m⁶A modification in plants.

Main Methods:

• Fused m⁶A writers (MTA/MTB) and eraser (ALKBH5) to catalytically dead Cas13a (dCas13a) to create programmable m⁶A editors.
• Applied editors to target specific RNA transcripts in *Nicotiana benthamiana* and *Arabidopsis thaliana*.
• Analyzed m⁶A level changes and off-target effects.

Main Results:

• Demonstrated efficient and specific deposition and removal of m⁶A on target RNA transcripts.
• Targeting SHORT-ROOT (SHR) transcripts increased m⁶A levels, promoted degradation, and enhanced plant growth.
• Observed increased plant height, biomass, leaf/root size, and grain weight in *Arabidopsis* with limited off-target effects.

Conclusions:

• Developed programmable m⁶A editing tools applicable to plant mRNA.
• These tools enable functional studies of individual m⁶A modifications in plants.
• Potential applications for crop improvement through targeted m⁶A editing.