Programmable RNA N⁶-methyladenosine editing by CRISPR-Cas9 conjugates.

Area of Science:

• Molecular Biology
• RNA Biology
• Epigenetics

Background:

• N⁶-methyladenosine (m⁶A) is a crucial RNA modification impacting post-transcriptional regulation.
• The functional impact of m⁶A modifications varies depending on their location within RNA molecules.
• Existing tools lack the precision to investigate the role of individual m⁶A sites.

Purpose of the Study:

• To develop a novel method for site-specific installation and removal of m⁶A modifications in cellular RNA.
• To enable functional studies comparing the effects of m⁶A at distinct RNA locations.
• To advance the understanding of the epitranscriptome's regulatory mechanisms.

Main Methods:

• Engineered CRISPR-Cas9 fused with an m⁶A methyltransferase ('m⁶A writers') for targeted RNA methylation.
• Developed CRISPR-Cas9 fusions with demethylases ALKBH5 or FTO ('m⁶A erasers') for site-specific demethylation.
• Utilized guide RNAs for precise targeting of m⁶A editing to specific RNA sequences.

Main Results:

• Demonstrated the ability to precisely install m⁶A modifications at specific sites in RNA without altering the primary sequence.
• Successfully achieved site-specific removal of m⁶A modifications using engineered demethylase fusions.
• Enabled functional comparisons of single m⁶A sites in different messenger RNA regions.

Conclusions:

• 'm⁶A editing' provides a powerful platform for RNA engineering and functional epitranscriptomic studies.
• This technology facilitates mechanistic investigations into the role of specific m⁶A modifications.
• Expands the toolkit for manipulating and studying RNA modifications in biological systems.