CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Type III-E CRISPR-Cas systems, featuring the Cas7-11 effector, offer precise RNA targeting for gene editing applications.
• Understanding the conformational dynamics of Cas7-11 is essential for optimizing its RNA editing potential.

Purpose of the Study:

• To investigate the conformational dynamics of the apo Cas7-11 effector protein.
• To elucidate the role of CRISPR RNA (crRNA) binding in regulating Cas7-11 structure and function.

Main Methods:

• Biochemical assays were employed to study Cas7-11.
• Amide hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) was used to probe protein dynamics.
• The effects of crRNA and target RNA binding on Cas7-11 conformation were analyzed.

Main Results:

• Apo Cas7-11 exhibits dynamic insertion sequences that fold upon crRNA binding.
• crRNA binding induces folding of catalytic loops and strengthens domain interactions, impacting processing and target cleavage.
• Mature crRNA binding leads to significant ordering of the insertion domain and conformational changes in TPR-CHAT binding sites.

Conclusions:

• crRNA binding is a critical regulator of Cas7-11 structure and function, influencing its RNA processing and editing capabilities.
• Cas7-11's ability to fully process CRISPR arrays and its target RNA cleavage efficiency are modulated by crRNA binding states.
• The study provides novel insights into the apo state and crRNA-mediated conformational changes of Cas7-11, paving the way for advanced RNA editing tools.