A versatile type VI CRISPR-based approach for targeted m⁶A demethylation in mRNAs

Area of Science:

• Epitranscriptomics
• Molecular Biology
• RNA Biology

Background:

• Epitranscriptomics studies RNA modifications, with N6-methyladenosine (m6A) being key in regulating mRNA functions.
• m6A is crucial for processes like spermatogenesis and DNA damage response, but targeted manipulation remains limited.

Purpose of the Study:

• To develop an efficient tool for site-specific m6A demethylation in human mRNAs.
• To provide a versatile platform for investigating m6A's functional roles.

Main Methods:

• Designed and constructed Dem6A-Vec, an "all-in-one" plasmid vector expressing a deactivated Cas13d-ALKBH5 fusion and a guide RNA.
• Utilized nanopore direct RNA sequencing to identify m6A sites in HeLa cells.
• Validated targeted demethylation using SELECT-qPCR on EEF2 and RRAGA genes.

Main Results:

• Dem6A-Vec successfully achieved site-specific m6A demethylation in target genes.
• Demonstrated the tool's precision and impact on mRNA stability.
• Showcased adaptability across various mRNA sites and methylation levels.

Conclusions:

• Dem6A-Vec offers a robust and scalable method for m6A research.
• This tool advances epitranscriptomic studies and holds potential for therapeutic applications.