Ba Cas12a3 represents a new subtype of type V CRISPR effector with collateral activity toward tRNA

Area of Science:

• Molecular Biology
• Microbiology
• Biochemistry

Background:

• CRISPR-Cas12 systems are RNA-guided nucleases with diverse antiviral mechanisms.
• Existing subtypes target DNA or RNA directly or induce abortive infections via collateral cleavage.

Purpose of the Study:

• To characterize a novel Type V CRISPR effector, BaCas12a3.
• To elucidate the unique tRNA-cleavage mechanism employed by BaCas12a3 for host growth inhibition.

Main Methods:

• Plasmid interference assays were used to assess host growth inhibition.
• Biochemical characterization of the BaCas12a3-crRNA ribonucleoprotein (RNP) complex.
• Cryo-electron microscopy (Cryo-EM) was employed to determine structural features.
• Mutagenesis studies were performed to analyze domain functions.

Main Results:

• BaCas12a3 inhibits host growth without triggering a DNA damage response, indicating a non-DNA double-strand break mechanism.
• Biochemical assays confirmed BaCas12a3 as an RNA-activating nuclease that specifically cleaves the 3' terminus of tRNAs.
• Cryo-EM structures revealed a bilobed architecture with a unique nucleic acid-loading (NL) domain adjacent to the RuvC catalytic center.
• Structural and mutagenesis analyses identified the NL domain and a zinc ribbon domain forming a gated substrate groove essential for specific tRNA recognition and cleavage.

Conclusions:

• BaCas12a3 utilizes a novel tRNA-cleavage mechanism for CRISPR immunity.
• The identified NL domain plays a critical role in tRNA recognition and substrate specificity.
• This finding broadens the understanding of CRISPR-Cas12 functional diversity and antiviral strategies.