Related Experiment Video
Updated: Apr 2, 2026
Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
Published on: October 18, 2022
Mechanistic basis for selective Csm6-2 activation by cyclic penta-adenylate in a type III CRISPR-Cas system
Ruyi Shi1, Mengquan Yang2,3, Yusong Liu2,3
1College of Chemistry, Fuzhou University, Fuzhou, China.
Abstract:
Type III CRISPR systems generate cyclic oligoadenylate (cOA, 3 to 6 AMPs) messengers upon detecting viral RNA, activating downstream effectors to defend against viral infection. Although cOA-activated effectors have been extensively characterized, the effectors specific to cA5-one of the most abundant cOA species produced during phage infection-have remained unexplored. Here, we report that the CRISPR ribonuclease Csm6 (Csm6-2) from Actinomyces procaprae selectively employs cA5 as its activator. Csm6-2 utilizes its HEPN domain, rather than the CARF domain, to mediate self-limiting cleavage of cOA activators. Cryo-EM structural analyses reveal that Csm6-2 functions as a homotetramer, and disruption of tetramer formation significantly reduces its ribonuclease activity. Although cA6 and cA5 bind Csm6-2 with comparable affinity, only cA5 induces CARF domain closure, stabilizes the tetramer, and remodels the active site in the HEPN domain. In contrast, the sixth AMP of cA6 imposes significant steric hindrance on CARF domain movement, preventing its closure and subsequent allosteric activation. These findings expand our understanding of the cOA signaling diversity and specific cOA recognition mechanisms in type III CRISPR immunity.
Insights
Type III CRISPR immunity uses cyclic oligoadenylate (cOA) messengers. Researchers discovered a specific cOA activator, cA5, for the Csm6 ribonuclease, revealing novel recognition mechanisms.
Area of Science:
- Molecular Biology
- Immunology
- Structural Biology
Background:
- Type III CRISPR systems utilize cyclic oligoadenylates (cOA) as signaling molecules to activate effector proteins against viral invaders.
- While many cOA-activated effectors are known, those specific to cA5, a common cOA during phage infections, remain uncharacterized.
Purpose of the Study:
- To investigate the unexplored effectors activated by cA5 in Type III CRISPR immunity.
- To elucidate the mechanism of specific cOA recognition and activation by CRISPR-associated enzymes.
Main Methods:
- Utilized Cryo-electron microscopy (Cryo-EM) for structural analysis.
- Performed biochemical assays to assess ribonuclease activity and ligand binding affinity.
- Investigated the role of different domains (HEPN, CARF) and oligomerization states (tetramer) in Csm6-2 function.
Main Results:
- Identified Csm6-2 from Actinomyces procaprae as a ribonuclease selectively activated by cA5.
- Cryo-EM revealed Csm6-2 functions as a homotetramer, with tetramer formation crucial for activity.
- Discovered that cA5 binding induces CARF domain closure and HEPN domain remodeling for activation, a process hindered by cA6 due to steric effects.
Conclusions:
- Csm6-2 represents a novel effector in Type III CRISPR immunity, specifically recognizing and being activated by cA5.
- The study reveals a unique allosteric activation mechanism involving cA5-induced conformational changes and tetramer stabilization.
- Findings expand the understanding of cOA signaling diversity and specific molecular recognition within CRISPR-Cas systems.
Related Concept Videos
02:53CRISPR and crRNAs
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
01:59CRISPR
01:59CRISPR
01:28CRISPR/Cas9 Genome Editing
01:23The Antiviral System of Bacteria and Archaea: CRISPR
01:28Global Regulatory Systems