In a groundbreaking study published in Nature, researchers have unveiled the detailed molecular mechanics by which the bacterial enzyme Ba1Cas12a3 executes its immune function through precise cleavage of tRNA molecules. This discovery sheds new light on the sophisticated strategies bacteria employ to defend themselves against viral invaders, expanding our understanding of the CRISPR-Cas systems beyond their traditional DNA-targeting roles.
Ba1Cas12a3, a member of the Cas12a family of nucleases, exhibits a unique mechanism of substrate recognition and cleavage triggered by RNA. The research team employed single-particle cryogenic electron microscopy (cryo-EM) to resolve the enzyme’s quaternary structure at an impressive resolution of 3.1 Å, capturing its interaction with three critical RNA components: crRNA, target RNA, and a specific transfer RNA — tRNA^Ala(UGC). This structural snapshot provides an exceptional view into how Ba1Cas12a3 orchestrates the cleavage of free tRNAs, a process integral to bacterial immunity.
The structural analysis revealed that Ba1Cas12a3 assembles into distinct lobes, reminiscent yet distinct from previously characterized Cas12a homologs. At its core, the enzyme contains a recognition (REC) lobe composed of REC1 and REC2 domains, and a nuclease (NUC) lobe including wedge (WED), PFS-interacting (PI), RuvC, zinc ribbon (ZR), and an insertion domain. Notably, a previously uncharacterized segment within the insertion domain spanning residues 855–960 exhibited no structural homologs in existing protein databases. This domain, designated the tRNA-loading domain (tRLD), plays a pivotal role in docking the tRNA substrate within the enzyme’s active site.
The research highlights two critical anchoring points facilitating tRNA capture. First, a small loop within the REC2 domain interacts specifically with the phosphate backbone of the tRNA’s T-arm through hydrogen bonding, establishing an initial recognition interface. Second, the acceptor stem and 3′ CCA tail of the tRNA are securely clamped by a collaboration between the tRLD and RuvC nuclease domain, positioning the scissile phosphate for cleavage. Detailed interactions include stacking of the terminal adenosine base between residues R902 and N924 and electrostatic stabilization from lysines K881 and K885, ensuring precise substrate positioning.
Interestingly, the 3′ hydroxyl group of the terminal adenosine projects into a cavity within the enzyme structure, a space hypothesized to accommodate the amino acid charged to the tRNA. Such structural accommodation suggests that Ba1Cas12a3 specifically targets free tRNAs not actively involved in translation, a selective cleavage strategy that minimizes collateral damage to the protein synthesis machinery during immune defense.
To probe the functional relevance of these structural features, the team performed an array of mutational analyses on both the tRNA substrate and Ba1Cas12a3 itself. Surprisingly, truncating the tRNA loops had negligible impact on cleavage efficiency, provided that the acceptor stem and 3′ CCA tail remained intact. A minimal tRNA mimic comprising just the anticodon loop, acceptor stem, and CCA tail was sufficient for cleavage, albeit with reduced binding affinity compared to the full-length tRNA. This underscores the enzyme’s adaptability in recognizing varied tRNA structures.
Further dissection of the CCA tail’s nucleotide composition revealed marked sensitivity to cytosine-to-guanine transversions, which significantly impaired cleavage, contrasting with a more tolerant response to cytosine-to-uracil transitions. Complementary studies on a related enzyme, Sm3Cas12a3, demonstrated even more stringent sequence specificity, highlighting divergent substrate recognition modalities within the Cas12a3 family. These findings emphasize how subtle nucleotide variations can critically modulate enzyme activity and specificity.
Attention then focused on mutagenesis within Ba1Cas12a3. Deletion of the tRLD domain, while not disrupting overall protein folding or binding to crRNA and target RNA, drastically diminished both in vitro cleavage activity and reporter gene silencing in transcription-translation (TXTL) assays. Targeted substitutions of residues involved in stabilizing the terminal adenosine (R902 and N924) similarly reduced immune function, establishing their essential role in substrate positioning. Intriguingly, a Y922A mutation paradoxically increased cleavage efficiency on truncated tRNA substrates but not on arbitrary RNA sequences, suggesting a finely tuned mechanistic involvement of this residue in modulating enzyme activity.
Mutations in the REC2 loop, responsible for T-arm recognition, impaired cleavage of full-length tRNAs but had limited effect on truncated substrates lacking the T-arm. This finding supports the mechanistic model where interactions with the T-arm assist in orienting entire tRNA molecules during cleavage, but are dispensable for minimal substrates. Collectively, these mutational insights reveal a sophisticated interplay between shape complementarity and charge-based interactions that orchestrate substrate selection and catalytic cleavage by Ba1Cas12a3.
This study challenges conventional wisdom by demonstrating that Ba1Cas12a3 exerts immunity through targeted cleavage of tRNA tails rather than direct DNA targeting, as seen in traditional CRISPR systems. By selectively incapacitating free tRNAs, the enzyme effectively disrupts bacterial and viral protein synthesis, halting infection progression. This RNA-triggered cleavage mechanism broadens our understanding of the diverse functional repertoire within CRISPR-Cas systems and illustrates the evolutionary ingenuity of bacterial immunity.
The discovery of the tRLD as a novel folding motif crucial for tRNA engagement opens exciting avenues for bioengineering. Harnessing this domain or its molecular principles could inspire the design of programmable RNA-targeting tools with applications ranging from synthetic biology to therapeutics. Moreover, the unique structural distinctions between Ba1Cas12a3 and other Cas12a homologs underscore the hidden diversity of CRISPR effectors yet to be explored.
Importantly, the finding that Ba1Cas12a3 targets the same tRNA region bound by the elongation factor Tu — a key player in delivering aminoacyl-tRNAs to the ribosome — suggests that the enzyme specifically cleaves translation-incompetent tRNAs, thereby fine-tuning the immune response without completely abolishing protein synthesis. This nuanced targeting strategy likely reflects evolutionary pressures to balance effective defense with cellular viability.
In conclusion, the comprehensive structural and functional characterization of Ba1Cas12a3 presented in this seminal work provides a transformative perspective on bacterial immune mechanisms. By elucidating how RNA-triggered nucleases can selectively cleave tRNA substrates, the study lays the foundation for innovative applications in biotechnology and offers a vivid example of nature’s molecular inventiveness in the microbial arms race.
Subject of Research: Ba1Cas12a3 enzyme mechanism in bacterial immunity and tRNA tail cleavage.
Article Title: RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity.
Article References:
Dmytrenko, O., Yuan, B., Crosby, K.T. et al. RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity. Nature (2026). https://doi.org/10.1038/s41586-025-09852-9
Image Credits: AI Generated
DOI: https://doi.org/10.1038/s41586-025-09852-9
Keywords: Ba1Cas12a3, CRISPR-Cas12a, tRNA cleavage, bacterial immunity, cryo-EM structure, RNA-guided nucleases, tRNA-loading domain, RuvC nuclease, translation disruption, structural biology
