RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity.

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Transfer RNAs (tRNAs) are crucial for translating genetic information and are recognized targets of innate immunity due to their role in suppressing viral replication.
• Bacterial CRISPR-Cas systems provide adaptive immunity against foreign genetic elements.

Purpose of the Study:

• To identify and characterize novel CRISPR-Cas nucleases involved in bacterial defense mechanisms.
• To elucidate the mechanism by which these nucleases target and inactivate tRNAs.
• To explore the potential applications of these nucleases in diagnostics and biotechnology.

Main Methods:

• Cell-based assays
• Biochemical assays
• Direct RNA sequencing
• Cryogenic electron microscopy (Cryo-EM)
• Design of synthetic RNA reporters

Main Results:

• Discovery of Cas12a3 effector nucleases from previously unreported type V CRISPR-Cas clades.
• Cas12a3 recognizes target RNA via CRISPR RNA and cleaves the 5'-CCA-3' tail of diverse tRNAs.
• This tRNA inactivation leads to growth arrest and anti-phage defense in bacteria.
• Cryo-EM structures reveal a unique tRNA-loading domain facilitating tRNA tail cleavage.
• Synthetic reporters demonstrate expanded capabilities for multiplexed RNA detection using CRISPR technology.

Conclusions:

• Cas12a3 nucleases employ a novel CRISPR-based immune strategy of tRNA inactivation for bacterial defense.
• This mechanism broadens the understanding of CRISPR-Cas functions beyond DNA targeting.
• The findings enhance the CRISPR toolbox for RNA detection and diagnostics.