Locus-specific detection of pseudouridine with CRISPR-Cas13a

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Pseudouridine (Ψ) is the most abundant RNA modification, crucial for RNA structure and function.
• Accurate identification of pseudouridine sites is essential for understanding RNA biology and disease.
• Current methods for pseudouridine detection can be complex or lack specificity.

Purpose of the Study:

• To develop a novel, efficient, and simple method for precise pseudouridine site identification.
• To validate targeted pseudouridine sites in various RNA types.

Main Methods:

• Combined the CRISPR-Cas13a system with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) chemical labeling.
• Utilized fluorescence measurement for Ψ site detection.
• Applied the method to ribosomal RNA (rRNA), messenger RNA (mRNA), and small nuclear RNAs (snRNA).

Main Results:

• Achieved precise identification of pseudouridine sites at specific loci within rRNA, mRNA, and snRNA.
• Demonstrated good efficiency and simplicity of the developed approach.
• Provided a straightforward and fast validation for targeted pseudouridine sites.

Conclusions:

• The developed CRISPR-Cas13a and CMC labeling method offers a powerful tool for pseudouridine site mapping.
• This technique facilitates rapid and accurate analysis of RNA modifications.
• Enables deeper insights into the functional roles of pseudouridylation in RNA.