# Structural and functional insights into the adenosine deaminase of the type III-B CRISPR–Cas system

## TL;DR

This study reveals how two CRISPR effectors, CAAD and Csx1, work together in a bacterial immune system to fight viral infections using a signaling molecule called cOA.

## Contribution

The paper introduces a dual-targeting mechanism involving CAAD and Csx1 for coordinated antiviral defense in type III-B CRISPR systems.

## Key findings

- CAAD and Csx1 are co-activated by cOA to degrade viral RNA and disrupt nucleotide metabolism.
- cA4/cA6 binding stabilizes CAAD hexamers and triggers ATP deamination.
- Both CAAD and Csx1 can degrade cA4, enabling cross-regulation and immune response termination.

## Abstract

Type III CRISPR–Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) systems confer antiviral immunity via cyclic oligoadenylate (cOA) signaling. Here, we elucidate a cooperative bacterial defense strategy involving two cOA-activated CRISPR-associated Rossmann fold (CARF)-containing effectors, adenosine deaminase CAAD and ribonuclease Csx1, in Thermoanaerobaculum aquaticum. Genomic analyses indicate widespread co-occurrence of CRISPR-associated adenosine deaminase (CAAD) with ancillary CARF-containing effectors in type III CRISPR systems, suggesting that multiple CARF-containing proteins may contribute to a coordinated cOA-dependent defense. Biochemical and structural studies reveal the intrinsic dynamics of CAAD hexamer, and demonstrate that cA4/cA6 binding stabilizes CAAD hexamers, triggering metal-ion-dependent conversion of ATP into inosine triphosphate. Concurrently, the downstream Csx1 is exclusively activated by cA4 to cleave single-stranded RNA. Strikingly, we found that both effectors are capable of degrading cA4, suggesting that this CAAD–Csx1 pair may be cross-regulated and achieve immunity through a dual-targeting mechanism: in response to infection, Csx1 degrades viral RNA while CAAD disrupts nucleotide metabolism via ATP deamination, which can be relieved via cA4 degradation when infection has been eliminated. This study proposes an enhanced defense mechanism through coordinated activation and regulation of multiple CRISPR effectors by a single signaling molecule, unveiling unprecedented complexity in CRISPR immunoregulation.

Graphical Abstract